A nickel-based plant polyphenol coordination polymer artificial enzyme and its preparation method and application

Abstract

The present invention discloses a nickel-based polyphenol coordination polymer artificial enzyme and its preparation method and application, belonging to the field of biomedical technology. Salvianolic acid B is dissolved in an organic solvent, dodecanol is added, and sulfuric acid is used as a catalyst, and heating reaction is carried out to obtain a salvianolic acid B esterified product; the salvianolic acid B esterified product is dissolved in an organic solvent, an aqueous solution of nickel acetate is added and mixed evenly to obtain a mixed solution, the mixed solution is ultrasonically degassed in a protective atmosphere, and then N-methylpyrrolidone is added, and heating reaction is carried out to obtain a black powdery solid Ni-SalB, which is the nickel-based polyphenol coordination polymer artificial enzyme. The present invention uses salvianolic acid B esterified product and nickel as structural units to prepare the 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, reducing apoptosis, and has good biocompatibility and no toxic side effects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a nickel-based plant polyphenol coordination polymer artificial enzyme, a preparation method thereof, and an application thereof. Background Art

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

[0003] The pathogenesis of SIMD involves the production of excessive reactive oxygen species (ROS), driving a vicious cycle of oxidative stress, lipid peroxidation, and inflammatory cascades. Although the endogenous antioxidant system (SOD, CAT, GPx) maintains redox homeostasis, their activity during sepsis decreases, allowing the accumulation of ROS. Specifically, the excessive accumulation of ROS exceeds the endogenous antioxidant system, triggering a pathological cascade including lipid peroxidation, genomic instability, and proteotoxic stress, ultimately leading to dysregulation. This oxidative cascade further activates redox-sensitive transcription factors, upregulating adhesion molecules and pro-inflammatory cytokines, thereby amplifying leukocyte infiltration and establishing a self-sustaining inflammatory microenvironment, accelerating myocardial remodeling. Given this dual pathogenic mechanism targeting both oxidative and inflammatory pathways simultaneously, current methods for treating sepsis-induced myocardial injury face significant limitations. Therefore, the development of innovative therapeutic drugs that can neutralize the excessive production of ROS and regulate the lesions is an important frontier area for the treatment of SIMD.

[0004] Traditional treatment regimens include radical scavengers (such as ascorbic acid, α-tocopherol) and broad-spectrum COX inhibitors (such as non-steroidal anti-inflammatory drugs), but these drugs face serious pharmacological limitations in terms of biocompatibility and pharmacokinetics. Although natural enzymes have significant catalytic activity and substrate specificity, they have encountered some limitations that hinder their clinical application, including high production and storage costs, biological instability, limited recyclability, and potential immunogenicity. To address these challenges, recent advances in artificial catalysts have demonstrated great potential in mitigating excessive 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, direct synthesis, tunable structural properties, and enhanced biological stability, making them a viable alternative 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 and can mimic the structural and functional properties of natural enzymes. Their unique features, including multiple catalytic sites, optimal size, inherent biocompatibility, and biodegradability, make MOCPs widely applicable to therapeutic interventions for various diseases. Although the 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 largely relies on multi-component strategies. These methods usually involve integrating different functional elements into a single nanoparticle, which often leads to complex design and synthesis processes, thus hindering scalability and large-scale production.

[0005] So far, various nanomaterials with enzyme-like activities have been developed, including metal oxides, metal nanoparticles, and polymers, which have become the focus of research due to their excellent physicochemical properties, biosecurity, and ability to inhibit ROS. Nanozymes are more stable and cheaper than natural enzymes. Currently, various nanostructures with the ability to catalyze ROS elimination or neutralization have been reported; for example, cerium oxide-zirconium oxide nanozymes and cobalt-doped carbon nanozymes (Co / PMCS) have successfully mimicked antioxidant enzymes such as SOD, catalase, and glutathione peroxidase, and have shown effective therapeutic effects in sepsis. Therefore, it is of great significance to develop nanomaterials with enzyme-like activities that have simple preparation methods. Polyphenols have attracted much attention in the biomedical field due to their antioxidant, anti-inflammatory, metabolic regulation, and neuroprotective properties. However, the coordination ability of their phenolic hydroxyl groups is weak, and they are prone to depolymerization, resulting in rapid metabolism of active molecules, short action time, functional attenuation, and low biological effects after in vivo metabolism. Improving the low in vivo bioavailability of polyphenols has become the main limitation of their application. To address these limitations, a safe and multifunctional metal-organic coordination polymer nanozyme needs to be designed to improve the catalytic performance of artificial nanozymes and enhance bioavailability. Thus, by inhibiting oxidative stress, inhibiting the release of inflammatory factors, reducing apoptosis, effectively alleviating myocardial injury, and effectively treating septic myocardial injury. Summary of the Invention

[0006] In view of the above-mentioned prior art, the object of the present invention is to provide a nickel-based plant polyphenol coordination polymer artificial enzyme, its preparation method, and application. The present invention uses salvianolic 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 apoptosis, and has good biocompatibility and no toxic side effects.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, a preparation method of a nickel-based plant polyphenol coordination polymer artificial enzyme is provided, including the following steps:

[0009] (1) Dissolve salvianolic acid B in an organic solvent, add dodecanol, and use sulfuric acid as a catalyst to heat and react to obtain salvianolic acid B ester;

[0010] (2) Dissolve the salvianolic acid B ester in an organic solvent, add an aqueous solution of nickel acetate and mix evenly to obtain a mixed solution. The mixed solution is ultrasonically degassed in a protective atmosphere, and then N-methylpyrrolidone is added, and heated and reacted to obtain a black powdery solid Ni-SalB, which is a nickel-based plant polyphenol coordination polymer artificial enzyme.

[0011] Preferably, in step (1), the organic solvent is tetrahydrofuran; the molar ratio of salvianolic acid B to dodecanol is 1:3; the volume of sulfuric acid accounts for 4% of the volume of the organic solvent; the concentration of sulfuric acid is 98 wt%.

[0012] Preferably, in step (1), the temperature of the heating reaction is 60 - 65 °C, and the time of the heating reaction is 5 - 10 days.

[0013] Preferably, in step (2), the organic solvent is dimethyl sulfoxide; the ratio of the amounts of the salvianolic acid B ester, nickel acetate, and N-methylpyrrolidone added is 1 mmol: 2 mmol: 1.6 mL.

[0014] Preferably, in step (2), the protective atmosphere is nitrogen; the number of times of ultrasonic degassing is 3 times, and each time is 3 min.

[0015] Preferably, in step (2), the temperature of the heating reaction is 85 °C, and the time of the heating reaction is 48 h.

[0016] In the second aspect of the present invention, there is provided a nickel-based plant polyphenol coordination polymer artificial enzyme obtained by the above preparation method.

[0017] In the third aspect of the present invention, there is provided the use of the nickel-based plant polyphenol coordination polymer artificial enzyme in the preparation of a drug for treating septic cardiomyopathy.

[0018] Preferably, the septic cardiomyopathy is lipopolysaccharide-induced septic cardiomyopathy.

[0019] Preferably, the concentration of the nickel-based plant polyphenol coordination polymer artificial enzyme is 100 μg / mL.

[0020] The beneficial effects of the present invention:

[0021] (1) The metal-organic coordination polymer with salvianolic acid B ester and nickel as structural units in the present invention has a simple preparation method and reduces the preparation cost. And although the material contains heavy metals, the hemolysis rate is lower than 2%, the biocompatibility is good, and there are no toxic side effects.

[0022] (2) The nickel-based plant polyphenol coordination polymer artificial enzyme Ni-SalB prepared in the present invention has superoxide dismutase (SOD)-like, catalase (CAT)-like, and glutathione peroxidase (GSH-Px)-like activities. Ni-SalB can catalyze the conversion of reactive oxygen species (ROS) into hydrogen peroxide (H 2 O 2 ), and then hydrogen peroxide is converted into oxygen (O 2 ). Ni-SalB is a promising scavenger of ROS (including H 2 O 2, O 2 •- and ·OH) and reactive nitrogen species (RNS), has spectral antioxidant activity, while enhancing its transmembrane absorption ability, improving its lipid solubility and bioavailability. It inhibits oxidative stress, suppresses the release of inflammatory factors, reduces apoptosis, and alleviates LPS-induced septic cardiomyopathy. Description of the Drawings

[0023] Figure 1 : Related characterization spectra of Ni-SalB, where (a) XPS spectrum of Ni-SalB; (b) spectrum of Ni 2p; (c) spectrum of O1s; (d) spectrum of C 1s; (e) X-ray diffraction pattern of Ni-SalB;

[0024] Figure 2 : SEM and TEM of Ni-SalB, where (a) SEM of Ni-SalB at a scale of 1 μm; (b) SEM of Ni-SalB at a scale of 500 nm; (c) SEM of Ni-SalB at a scale of 200 nm; (d) TEM of Ni-SalB at a scale of 1 μm; (e) TEM of Ni-SalB at a scale of 50 nm; (f) HR-TEM of Ni-SalB at a scale of 10 nm; (g) elemental distribution map of Ni-SalB at a scale of 500 nm; (h) C elemental distribution map of Ni-SalB; (i) O elemental distribution map of Ni-SalB; (j) Ni elemental distribution map of Ni-SalB;

[0025] Figure 3 : Maps of Ni-SalB scavenging superoxide anion, hydroxyl radical, hydrogen peroxide and reactive nitrogen, where (a) UV-visible spectrum of O 2 •- produced by treating the AP-TEMED system with different concentrations of Ni-SalB (0, 25, 50, 75, 100 and 200 μg mL-1); (b) scavenging effect of Ni-SalB on O 2 •- ; (c) UV-visible spectrum of TMB treated with different concentrations of Ni-SalB; (d) scavenging effect of Ni-SalB on ·OH; (e) UV-visible spectrum of GSH and benzoic acid solution treated with Ni-SalB; (f) scavenging effect of Ni-SalB on H 2 O 2 ; (g) UV-visible spectrum of DPPH after treatment with Ni-SalB; (h) scavenging effect of Ni-SalB on DPPH; (i) O 2 •- and Ni-SalB + O 2•- ESR spectra of; (j) ESR spectra of OH and Ni-SalB+·OH; (k) ESR spectra of DPPH and Ni-SalB+DPPH;

[0026] Figure 4 : In vitro antioxidant 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, H 2 O 2 , Ni-SalB or both, scale bar 200 μm;

[0027] Figure 5 : Biocompatibility of Ni-SalB, where (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 hearts, livers, spleens, lungs and kidneys of mice in the PBS group and Ni-SalB group after 14 days;

[0028] Figure 6 : (a) Transmission electron microscopy images of mitochondria of mice in different groups; (b) Echocardiograms of mice in different groups; (c) H&E staining images of mice in different groups; (d) Masson images of mice in different groups; (e) Ejection fraction of mice in different groups; (f) Fractional shortening of the short axis of mice in different groups; (g) Left ventricular end-systolic volume of mice in different groups; (h) Left ventricular end-diastolic volume of mice in different groups; (i) Creatine kinase isoenzyme of mice in different groups; (j) Lactate dehydrogenase of mice in different groups; (k) Tumor necrosis factor-α of mice in different groups; (l) Interleukin 1-β of mice in different groups; (m) Interleukin-6 of mice in different groups; (n = 3, error bars represent standard deviation);

[0029] Figure 7 : (a) SOD levels of mice in each group; (b) Malondialdehyde 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;

[0030] Figure 8 : Synthetic route map of Ni-SalB. Detailed implementation manners

[0031] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0032] As introduced in the background art 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, functional attenuation, and low biological effects after in vivo metabolism. Improving the low bioavailability of polyphenols in vivo has become the main limitation of their application.

[0033] Based on this, the purpose of the present invention is to provide a preparation method of a nickel-based plant polyphenol coordination polymer artificial enzyme. First, the present invention uses salvianolic acid B and dodecanol to prepare a salvianolic acid B ester through an esterification reaction; then, using the salvianolic acid B ester and nickel as structural units, a metal-organic coordination polymer Ni-SalB is prepared by a one-step hydrothermal reaction.

[0034] Ni-SalB has integrated and efficient catalytic performance, and at the same time exhibits superoxide dismutase (SOD)-like and glutathione peroxidase (GSH-Px)-like activities. Specifically, it catalyzes the conversion of reactive oxygen species (ROS) into hydrogen peroxide (H 2 O 2 ), and then hydrogen peroxide is converted into water (H 2 O), while enhancing its transmembrane absorption ability, improving its liposolubility 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 reduces 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 reduces myocardial injury by inhibiting oxidative stress, inhibiting the release of inflammatory factors, and reducing apoptosis.

[0035] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific embodiments.

[0036] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels.

[0037] Example 1: Preparation of Ni-SalB

[0038] (1) Dissolve salvianolic acid B (1 mmol) in tetrahydrofuran (3 mL), then add dodecanol (3 mmol), use sulfuric acid (98 wt%, 0.12 mL) as a catalyst, heat at 65 °C for 5 days, monitor the reaction progress by reverse-phase HPLC, terminate the reaction by adding 2 mL of sodium carbonate (1 M), then dissolve the mixture in 100 mL of heated acetonitrile and filter, and store at 4 °C until purification to obtain the salvianolic acid B ester.

[0039] (2) Dissolve the salvianolic acid B ester (36 mg) in 8 mL of DMSO to obtain an ester solution. Dissolve 25 mg of Ni(OAc) 2 ·4H 2 O in 16 mL of deionized water to obtain a Ni(OAc) 2 ·4H 2 O solution. Add the Ni(OAc) 2 ·4H 2 O solution to the ester solution and stir evenly to obtain a mixed solution. The mixed solution is degassed by ultrasonic treatment (40 kHz, 300 W) for 10 minutes under nitrogen protection. Subsequently, add 1.6 mL of N-methylpyrrolidone (NMP) to the mixed solution and continuously stir at 85 °C for 48 hours. After cooling, centrifuge to collect the crude product, soak it in DMF overnight to remove residual impurities, wash it thoroughly with deionized water, and finally freeze-dry to obtain Ni-SalB. The specific synthesis route is shown in Figure 8 .

[0040] Comparative Example 1: Preparation of Ni-SalB-NE

[0041] Dissolve salvianolic acid B (36 mg) in 8 mL of DMSO to obtain a salvianolic acid B solution. Dissolve 25 mg of Ni(OAc) 2 ·4H 2 O in 16 mL of deionized water to obtain a Ni(OAc) 2 ·4H 2 O solution. Add the Ni(OAc) 2 ·4H 2 O solution to the salvianolic acid B solution and stir evenly to obtain a mixed solution. The mixed solution is degassed by ultrasonic treatment (40 kHz, 300 W) for 10 minutes under nitrogen protection. Subsequently, add 1.6 mL of N-methylpyrrolidone (NMP) to the mixed solution and continuously stir at 85 °C for 48 hours. After cooling, centrifuge to collect the crude product, soak it in DMF overnight to remove residual impurities, wash it thoroughly with deionized water, and finally freeze-dry to obtain Ni-SalB-NE.

[0042] Example 2: Characterization of Ni-SalB prepared in Example 1

[0043] (1)Determination of X-ray photoelectron spectroscopy of Ni-SalB: The composition of Ni-SalB was studied by X-ray photoelectron spectroscopy. As Figure 1 shown in (a), the full-scan XPS spectrum of Ni-SalB confirmed the presence of carbon (C), oxygen (O), and nickel (Ni). Figure 1 (b) High-resolution analysis of the Ni 2p spectrum showed two characteristic peaks 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 (c) The O1s spectrum in (c) had four distinct peaks at 531.0 eV, 531.8 eV, 532.6 eV, and 533.5 eV, corresponding to Ni-O, C-O, O-H, and C=O bonds, respectively. In addition, Figure 1 (d) The C 1s spectrum in (d) had three peaks at 284.8 eV, 286.4 eV, and 288.5 eV, which were assigned to C-C, C-O, and C=O bonds, respectively, further confirming the successful coordination between Ni and salvianolic acid B (SalB). Based on these results, it can be concluded that salvianolic acid B and nickel metal ions were successfully coordinated to form a stable Ni-SalB metal-organic coordination polymer.

[0044] (2) Understanding the morphology of Ni-SalB through X-ray diffraction patterns, as Figure 1 shown in (e). X-ray diffraction showed only a broad peak around 25°, reflecting the amorphous structure of the synthesized Ni-SalB.

[0045] (3) Observing the morphology of Ni-SalB through SEM and TEM, it can be seen from Figure 2 (a) to Figure 2 (d) that Ni-SalB exhibited an irregular structure, composed of stacked irregular near-spherical particles, with interconnected mesopores and macropores. Figure 2 High-resolution transmission electron microscopy (HR-TEM) analysis in (e) showed no aggregation of metal-based nanoparticles or lattice fringes, further confirming the amorphous nature of the synthesized material. Figure 2 Energy-dispersive X-ray spectroscopy and elemental mapping results in (f) showed that carbon (C 56.69%), oxygen (O 33.68%), and nickel (Ni 9.63%) were uniformly distributed throughout the carbon-based porous framework.

[0046] (4)Detect the scavenging ability of Ni-SalB against reactive oxygen species and reactive nitrogen species. Use a superoxide anion scavenging ability detection kit (Slorbio, BC1410) to detect the O 2 •- scavenging ability of Ni-SalB at different concentrations (0, 25, 50, 75, 100, and 200 μg / mL). According to the instructions of the superoxide anion scavenging ability detection kit, use the AP-TEMED system to generate superoxide anions, which then react with hydroxylamine hydrochloride to produce nitrite ions (NO 2 - ). These nitrite ions interact with sulfanilamide and α-naphthylamine to form a red azo compound. This compound has a characteristic absorption peak at 530 nm. Operate according to the instructions of the kit, measure the absorbance values of the blank solution and the solution containing Ni-SalB at 530 nm, and record them as AB and AT. The calculation formula for the O 2 •- scavenging rate is: D% = (AB - AT) ÷ AB × 100%.

[0047] Utilize the AP-TEMED / O 2 •- hydroxylamine colorimetric system to explore the SOD mimetic activity. As shown in Figure 3 Figure (a) and Figure 3 Figure (b), Ni-SalB exhibits dose-dependent superoxide dismutase-like activity, achieving an O -1 scavenging efficiency of 82.4 ± 4.4% at 200 μg mL 2 •− .

[0048] (5)Use the 3,3′,5,5′-tetramethylbiphenylmethane (TMB) colorimetric method to detect the ·OH scavenging ability of Ni-SalB for the solution at different concentrations (0, 25, 50, 75, 100, and 200 μg / mL). Prepare a PBS buffer solution (0.5 M, pH 4.5) containing TMB (1.5 mM), hydrogen peroxide (10 mM), ferrous sulfate (0.5 mM), and different concentrations of Ni-SalB in the dark and let it stand for 5 min. Then centrifuge (10000 rpm, 2 min), measure the absorbance at 652 nm, and the ·OH scavenging rate is D% = (AB - AT) ÷ AB × 100%. Measure the absorbance values of the blank solution and the solution containing Ni-SalB at 652 nm and record them as AB and AT.

[0049] Figure 3 Figure (c) and Figure 3(d) The elimination ability of hydroxyl radicals was investigated using the Fenton reaction / TMB oxidation system. At 200 μg / mL, Ni-SalB exhibited the activity of scavenging ·OH, with an inhibition rate of 39.9 ± 2.0%, showing an obvious concentration-dependent behavior.

[0050] (6) Using a glutathione peroxidase activity assay kit (Shanghai Yuanye Bio-Technology Co., Ltd., R21877-50T), the glutathione peroxidase (GPx)-like activity of Ni-SalB was evaluated at different concentration ranges (0, 25, 50, 75, 100, and 200 μg / mL). Ni-SalB and benzoic acid chromogenic solution were added to the GSH working solution. The concentration of the yellow anion generated in the GSH and benzoic acid coloring solution was measured using an ultraviolet-spectrophotometer at 422 nm.

[0051] Then, the cascade catalytic ability of the GPx-like was investigated by the hydrogen peroxide decomposition method. As Figure 3 (e) and Figure 3 (f) showed, Ni-SalB also had glutathione-dependent peroxidase activity. In the presence of 1 mM GSH, the hydrogen peroxide scavenging rate was 49.9 ± 3.9% at 200 μg / mL, indicating the effective regeneration of the catalytic cycle.

[0052] (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). The 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 (10000 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 solution containing Ni-SalB were measured at 517 nm and recorded as AB and AT.

[0053] The RNS neutralization ability of Ni-SalB was evaluated by the DPPH radical scavenging test. As Figure 3 (g) and Figure 3 (h) showed, Ni-SalB also showed a dose-responsive RNS elimination ability, reaching 42.6 ± 9.8% at 200 μg / mL.

[0054] (8) The free radical scavenging mechanism of Ni-SalB was verified by electron spin resonance (ESR) spectroscopy. Hydroxyl radical (•OH) detection: In a phosphate buffer solution (0.5 M, 600 μL) at pH 4.5, through the Fenton reaction system (containing FeSO 4(1 mg / mL, 75 μL), H 2 O 2 (10 M, 75 μL)) to generate hydroxyl radicals, and the DMPO spin trap agent (3% v / v, 20 μL) was added. The ESR spectrum showed a characteristic quartet signal (intensity ratio 1:2:2:1), indicating the formation of a stable spin adduct of DMPO with •OH; Superoxide anion (O 2 •- ) detection: In phosphate buffer at pH 7.4, O 2 •- was generated by the photolysis reaction (4000 lux illumination) of riboflavin (20 μM) and L-methionine (13 mM). After being trapped by DMPO, a unique sextet signal was formed, indicating the presence of superoxide radicals; DPPH radical scavenging experiment, using DPPH ethanol solution as the radical source; After treatment with Ni-SalB (200 μg / mL), the attenuation of the ESR signal quantitatively indicated its ability to scavenge ROS and RNS.

[0055] To further evaluate the scavenging ability of Ni-SalB on reactive oxygen and nitrogen species (RONS), electron spin resonance (ESR) spectroscopy was employed, which provided a direct visualization of the reduction of radical signals. As Figure 3 shown in (j), DMPO spin trapping showed a 1:2:2:1 multiplet pattern caused by ·OH. Notably, in the presence of Ni-SalB, the signal intensity of ·OH was significantly reduced (the intensity decreased by 74.4% at 200 μg / mL), indicating the high sensitivity of Ni-SalB to ·OH scavenging. Similarly, Figure 3 the addition of Ni-SalB in (i) led to a significant decrease in the peak intensity of O 2 •- , while the signal of the DEPMPO-O 2 •- adduct decreased by 78.2%, confirming its effective consumption of O 2 •- species. In addition, Figure 3 (k) showed that in the presence of Ni-SalB, the ESR signal peak of DPPH radicals was significantly weakened (the signal decreased by 79.8% at 200 μg / mL), showing its strong DPPH scavenging ability. These results together indicate that Ni-SalB has multiple antioxidant properties and can effectively remove ROS and RNS.

[0056] Test Example 1: In vitro antioxidant stress test of Ni-SalB and its protective effect on cardiomyocytes

[0057] (1) ROS staining of LPS-induced H9C2 cardiomyocytes: H9C2 cardiomyocytes (rat cardiomyocytes, Chinese Academy of Sciences Cell Bank) 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.

[0058] Given the good enzymatic activity of Ni-SalB in scavenging reactive species, its intracellular antioxidant capacity and cytoprotective effects were further investigated. Lipopolysaccharide (LPS) is a known bacterial endotoxin that stimulates the production of intracellular reactive oxygen species (ROS) and was used to induce oxidative stress in H9C2 cardiomyocytes. After LPS treatment, the ROS level in H9C2 cells increased significantly, as demonstrated by the increased green fluorescence intensity detected using the ROS-sensitive probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). Notably, treatment with Ni-SalB (100 μg / mL) effectively scavenged the overproduced ROS, reducing the fluorescence intensity to a level comparable to that of untreated cells, as shown in 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.

[0059] (2) Live / dead staining of H 2 O 2 -induced oxidative stress in cardiomyocytes: H9C2 cells were seeded in 6-well plates and incubated for 24 h,

[0060] and the cells were treated with H 2 O 2 (700 μM), Ni-SalB (100 μg / mL), and H 2 O 2 +Ni-SalB, respectively. After incubation, Calcein / PI solution was added to the cardiomyocytes and incubated for another 30 min. The stained cells were observed under a fluorescence microscope.

[0061] In addition, the cytoprotective effect of Ni-SalB on H9C2 cardiomyocytes was evaluated under oxidative stress induced by hydrogen peroxide (H 2 O 2 ). The live / dead cell staining assay showed that treatment with hydrogen peroxide (H 2 O 2 ) significantly increased cell death, indicating the presence of PI-positive cells (red fluorescence). Notably, Figure 4 pretreatment with Ni-SalB completely prevented H2 O 2 Induced cell death. These results highlight that Ni-SalB has special antioxidant activity, which can effectively protect cells from oxidative stress-induced damage by neutralizing intracellular ROS.

[0062] Experimental Example 2: In vitro biocompatibility study of Ni-SalB

[0063] (1) Hemolysis experiment

[0064] Fresh blood of 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, washed with PBS, centrifuged again, and repeated 3 times. Finally, red blood cells were taken to form a 5% red blood cell suspension. The red blood cells were mixed with Ni-SalB at different concentrations (25 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL, 150 μg / mL) at a ratio of 1:1 (volume ratio of red blood cell suspension to Ni-SalB at different concentrations) and incubated at 37 °C for 3 h. After centrifugation, the supernatant was taken to measure the OD value (540 nm). 0.5 mL of diluted red blood cell suspension and 0.5 mL of PBS were used as negative control, and 0.5 mL of diluted red blood cell suspension and 0.5 mL of distilled water were used as positive control. The hemolysis rate was calculated using the following formula:

[0065] Hemolysis volume (%) = (As - An) / (Ap - An) × 100%;

[0066] Where "As" is the absorbance obtained from the supernatant after adding Ni-SalB to red blood cells. "An" is the absorbance obtained from the supernatant after adding PBS to red blood cells (negative control). "Ap" is the absorbance obtained from the supernatant after adding distilled water to red blood cells (positive control).

[0067] As Figure 5 As shown in (a) and 5(b), within the concentration range showing antioxidant activity, Ni-SalB showed only a small amount (less than 2%) or no hemolytic 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%. 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.

[0068] (2) Cytotoxicity experiment

[0069] The cytotoxicity of Ni-SalB was studied using rat cardiomyocytes H9C2 (from the Cell Bank of the Chinese Academy of Sciences). H9C2 cells were seeded in 96-well plates at a density of 5×10 3 cells per well and then incubated with different concentrations of Ni-SalB solution for 24 h. After adding 10 µL of CCK-8 working solution and incubating for 30 min, the OD value at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. Each experiment was repeated three times.

[0070] As Figure 5 (c) shows the cell viability of H9C2 rat cardiomyocytes cultured with different concentrations of Ni-SalB (0 - 150 μg / mL) for 2 days. It can be clearly seen that as the concentration of the material decreased, the relative cell viability gradually increased, and its value remained greater than 90% at all experimental concentrations, indicating no toxicity to H9C2 rat cardiomyocytes. All these results confirmed good biocompatibility in vitro.

[0071] Test Example 3: In Vivo Biocompatibility Study

[0072] Ni-SalB (100 μg / mL) was intraperitoneally injected into C57BL / 6 male mice (8 weeks old, purchased from Shandong Pengyue Laboratory Animal Technology Co., Ltd.) to evaluate its in vivo biocompatibility; an equal volume of PBS was injected as a control. Fourteen days after administration, the major organs, including the heart, liver, spleen, lungs, and kidneys, were collected and subjected to histopathological examination by hematoxylin and eosin (H&E) staining. As Figure 5 (d) shows, no signs of tissue inflammation, lesions, or other abnormalities were observed in any of the organs in the Ni-SalB treatment group, confirming its good in vivo biocompatibility. Taken together, these findings indicate that Ni-SalB is a highly biocompatible material suitable for biomedical applications.

[0073] Test Example 4

[0074] (1) In Vivo Study of Lipopolysaccharide (LPS)-Induced Septic Cardiomyopathy:

[0075] Based on the multi-enzyme mimicking ability and good biocompatibility of Ni-SalB, its therapeutic potential for lipopolysaccharide-induced septic cardiomyopathy was systematically investigated through multi-modal evaluations. Eight-week-old male C57BL / 6 mice were used and divided into 5 groups (n = 6), namely the PBS group, the Ni-SalB group, the LPS group, the LPS+Ni-SalB group, and the LPS+Ni-SalB-NE group. In the Ni-SalB group and the LPS+Ni-SalB group, Ni-SalB was intraperitoneally injected in advance to verify its protective effect on septic cardiomyopathy. In the LPS+Ni-SalB-NE group, Ni-SalB-NE was intraperitoneally injected in advance to verify its protective effect on septic cardiomyopathy. The injection dose was 100 μg / mL for all. The PBS group was intraperitoneally injected with an equal volume of PBS in advance, and the LPS group was not injected. The administration (injection) was carried out continuously for 14 days. Then, the LPS group, the LPS+Ni-SalB group, and the LPS+Ni-SalB-NE group were intraperitoneally injected with LPS (12 mg / kg) to establish an LPS-induced septic cardiomyopathy model. After 24 hours, echocardiography was performed to determine the establishment of the septic cardiomyopathy model. Then, the blood of the mouse heart and the heart, liver, spleen, lungs, and kidneys were taken for biochemical and H&E staining, etc. to verify the in vivo effects.

[0076] As Figure 6 shown in (a), electron microscopy showed that the mitochondrial cristae of the mice in the LPS group were disrupted and the myofibrils were disordered. These changes were alleviated in the mice of the LPS+Ni-SalB group and the LPS+Ni-SalB-NE group, and the effect of the LPS+Ni-SalB group was better than that of the LPS+Ni-SalB-NE group. Meanwhile Figure 6 (b) and Figure 6 (e)~ Figure 6 (h) showed that compared with the PBS group, echocardiography showed signs of cardiac insufficiency in the mice of the LPS group. Among them, the fractional shortening (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 indicated lipopolysaccharide-induced in vivo cardiac injury and dysfunction. In contrast, compared with the LPS group, the FS and EF were significantly improved in the LPS+Ni-SalB group and the LPS+Ni-SalB-NE group. And the LPS+Ni-SalB group improved cardiac dysfunction more significantly. These results indicated that Ni-SalB could effectively alleviate lipopolysaccharide-induced myocardial dysfunction. In addition, the potential to alleviate the effect of lipopolysaccharide could also be demonstrated from the improvement of histopathology. As Figure 6As shown in Figures 6(c) and 6(d), H&E and Masson's trichrome staining showed that compared with the LPS group, the mice in the LPS+Ni-SalB group and the LPS+Ni-SalB-NE group showed less severe inflammatory responses, bleeding, structural disorders, and fibrosis. Meanwhile, 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 superior therapeutic effects. In addition, Figure 6 (i) and Figure 6 (j) showed that the levels of serum creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) in the LPS+Ni-SalB group and the LPS+Ni-SalB-NE group were significantly lower than those in the LPS group. Further comparison with the LPS+Ni-SalB-NE group indicated the superiority of Ni-SalB in protecting against LPS-induced cardiac injury. 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 damage and exacerbating cardiac dysfunction.

[0077] To further explore its anti-inflammatory effect, cardiac blood was extracted from the mice, and an ELISA kit (Servicebio GEM0002-96T / GEM0004-96T / GEM0001-96T) was 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-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in the serum of the mice in the LPS+Ni-SalB group and the LPS+Ni-SalB-NE group were significantly downregulated, and the effect of the LPS+Ni-SalB group was more obvious. This indicates that Ni-SalB can effectively reduce the inflammatory response by inhibiting the production of lipopolysaccharide-induced inflammatory cytokines IL-1β, TNF-α, and IL-6. Therefore, Ni-SalB shows good anti-inflammatory potential in the treatment of septic cardiomyopathy.

[0078] As previously reported, septic cardiomyopathy is also associated with increased oxidative stress. To evaluate this, specific kits (Servicebio G4306-96T / G4302-96T) were used to measure the activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) in the cardiac blood of the mice. Figure 7 (a)~ Figure 7(b) showed that compared with the LPS group, Ni-SalB and Ni-SalB-NE significantly increased SOD activity and decreased MDA levels. In addition, DHE (dihydroethidium) staining of heart 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 a significant increase in the generation of ROS (reactive oxygen species). However, according to Figure 7 (c), it can be seen that in the LPS+Ni-SalB and LPS+Ni-SalB-NE groups, compared with the LPS group, the intensity of the red fluorescence signal was significantly reduced, confirming that Ni-SalB can effectively inhibit the production of ROS in mouse myocardium. These results were consistent with the in vitro experimental results. In addition, Figure 7 Tunel staining in (d) showed that compared with the LPS group, LPS+Ni-SalB and LPS+Ni-SalB-NE significantly inhibited LPS-induced cardiomyocyte apoptosis. And Ni-SalB was superior to Ni-SalB-NE, indicating that Ni-SalB inhibits LPS-induced oxidative stress and cardiomyocyte apoptosis by increasing the level of antioxidant enzymes and reducing ROS generation indicators.

[0079] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included within 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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