Quercetin carbon dots with antibacterial and anti-inflammatory characteristics as well as preparation method and application of quercetin carbon dots

Abstract

The invention discloses a quercetin-based carbon dot with antibacterial and anti-inflammatory characteristics as well as a preparation method and application of the quercetin-based carbon dot. The preparation method of the quercetin-based carbon dots comprises the following steps: (1) S1, adding quercetin and 4-aminophenol into pure water, mixing, carrying out ultrasonic treatment, transferring the obtained mixed solution into a high-pressure reaction kettle, and reacting under heating; and (2) after the reaction is finished in the step (S2), cooling to room temperature, filtering, and freeze-drying filtrate to obtain the quercetin-based carbon dots (QA-CDs). The QA-CDs provided by the invention show a remarkable ROS (reactive oxygen species) removal capability and effectively regulate the expression level of inflammation-related factors; the QA-CDs can regulate the inflammation microenvironment while effectively exerting the antibacterial activity, so that the QA-CDs show obvious dual-function characteristics. A new scheme is established for application of flavone-based compounds, a promising research framework is provided for development of innovative bioactive materials with dual treatment effects, and development of innovative strategies in the field of bacterial infection treatment can be greatly promoted.

Description

Technical Field

[0001] The present invention relates to the fields of medicine and nanomaterials, and in particular to quercetin carbon dots with antibacterial and anti-inflammatory properties, and a preparation method and application thereof. Background Art

[0002] Public health emergencies caused by bacterial infections frequently occur in multiple areas, including food safety, healthcare, and environmental sanitation. Upon invading the host organism, bacteria release endotoxins that activate the innate immune system, thereby inducing a strong inflammatory response. In this pathological process, dysregulated secretion of inflammatory factors by immune cells (such as macrophages) leads to abnormally elevated levels of cytokines in the circulation (known as a "cytokine storm"), which in turn leads to overactivation of the immune system. This disruption of immune homeostasis ultimately contributes to the pathogenesis of various inflammatory-related diseases, including secondary viral infections, autoimmune diseases, sepsis, and malignancies, significantly increasing the morbidity and mortality of related diseases. Antibiotics, as the main treatment for bacterial infections, have significantly reduced their therapeutic efficacy due to widespread use and the spread of animal-derived resistance genes, leading to the widespread emergence of multidrug-resistant strains. This critical situation requires the development of new antimicrobial agents to address the escalating threat of bacterial infections.

[0003] In recent years, breakthrough advances in nanomaterials have provided innovative solutions for antimicrobial and anti-inflammatory therapies. Among them, carbon-based nanomaterials have attracted considerable interest due to their excellent biocompatibility, tunable physicochemical properties, and multifaceted antimicrobial mechanisms. The unique advantages of these materials stem from the rich surface functional groups within their core-shell structures, which not only endow carbon dots (CDs) with enzyme-like catalytic activity but also enable specific recognition and response to biomolecules, thereby precisely regulating targeted biological responses. Phenolic compounds, including aminophenols and their derivatives, have been widely used in the antimicrobial field due to their remarkable antimicrobial properties. These compounds possess a characteristic benzene ring structure with hydroxyl (-OH) functional groups, which can strongly interact with microbial cell membranes. This interaction leads to structural disruption of microbial cells, effectively inhibiting their growth and proliferation. Studies have shown that 4-aminophenol-modified gold nanoparticles exhibit significant antimicrobial activity by specifically binding to bacterial 16S rRNA and disrupting cell wall integrity. Many bioactive substances have attracted increasing attention due to their remarkable antimicrobial and anti-inflammatory properties. Quercetin (QU) is an important flavonoid compound that exhibits diverse pharmacological activities in the biomedical field. QU has diverse biological activities, including anticancer, antioxidant, cardioprotective, anti-inflammatory, and neuroprotective effects. However, the presence of multiple phenolic hydroxyl groups and conjugated systems in its molecular structure easily leads to cocrystal formation, resulting in limited bioavailability, poor water solubility, and chemical instability. To overcome these limitations, researchers have proposed a strategy to combine QU with carbon-based nanomaterials. In recent years, various QU-derived CDs have been successfully developed through green synthetic methods such as hydrothermal synthesis. Studies have shown that QU-derived CDs exhibit unique advantages in inhibiting β-amyloid fibrillization in Alzheimer's disease, developing novel full-spectrum UV absorbers, and biosensing applications (such as dopamine detection in serum). These findings indicate that CDs derived from QU not only have improved physicochemical properties but also possess novel biological functions. However, current research on CDs derived from bioactive precursors has mainly focused on the unilateral evaluation of antimicrobial or anti-inflammatory effects, lacking a comprehensive strategy for dual treatment of acute inflammatory microenvironments. Nonetheless, nanosystems still suffer from limitations such as complex synthetic processes and single modes of action, highlighting the urgent need to develop novel formulations that combine inflammation-modulating functions with broad-spectrum antimicrobial efficacy. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies in the above-mentioned prior art and provide a quercetin carbon dot with antibacterial and anti-inflammatory properties, a preparation method thereof and an application thereof.

[0005] To solve the above technical problems, the present invention adopts a technical solution: a method for preparing quercetin-based carbon dots with antibacterial and anti-inflammatory properties, comprising the following steps:

[0006] S1: quercetin and 4-aminophenol were added to pure water, mixed, and sonicated. The resulting mixed solution was transferred to a high-pressure reactor and heated for reaction;

[0007] S2: After the reaction is completed, the mixture is cooled to room temperature, filtered, and the filtrate is freeze-dried to obtain quercetin-based carbon dots.

[0008] Preferably, the reaction temperature in step S1 is 180-220° C., and the reaction time is 4-16 hours.

[0009] Preferably, step S1 is specifically: adding 0.1-0.4 g of quercetin and 0.1-0.4 g of 4-aminophenol to 20-40 mL of pure water, mixing thoroughly, and then ultrasonically treating for 5-20 minutes, transferring the resulting mixed solution to a polytetrafluoroethylene-lined autoclave, and reacting at 180-220° C. for 4-16 hours.

[0010] Preferably, step S2 is specifically as follows: after the reaction is completed, the mixture is naturally cooled to room temperature, the product is preliminarily filtered using filter paper, and then finely filtered through a 0.2-0.3 μm membrane filter, and finally, the filtrate is freeze-dried to obtain quercetin-based carbon dots.

[0011] Preferably, the method for preparing quercetin-based carbon dots with antibacterial and anti-inflammatory properties comprises the following steps:

[0012] S1: 0.2 g of quercetin and 0.2 g of 4-aminophenol were added to 40 mL of pure water, mixed thoroughly, and then ultrasonicated for 10 minutes. The resulting mixed solution was transferred to a polytetrafluoroethylene-lined autoclave and reacted at 200 °C for 8 hours;

[0013] S2: After the reaction is completed, the mixture is naturally cooled to room temperature, and the product is preliminarily filtered using filter paper, and then finely filtered through a 0.22 μm membrane filter. Finally, the filtrate is freeze-dried to obtain quercetin-based carbon dots, which are stored at 4°C for future use.

[0014] The present invention also provides quercetin-based carbon dots, which are prepared by the method described above.

[0015] The present invention also provides a use of the quercetin-based carbon dots as an antibacterial agent.

[0016] The present invention also provides a use of the quercetin-based carbon dots described above in the preparation of an antibacterial composition or an antibacterial drug.

[0017] The present invention also provides a use of the quercetin-based carbon dots as an anti-inflammatory preparation.

[0018] The present invention also provides a use of the quercetin-based carbon dots described above in the preparation of an anti-inflammatory composition or an anti-inflammatory drug.

[0019] The beneficial effects of the present invention are:

[0020] To address the multifaceted challenges posed by inflammatory responses caused by bacterial infection and immune disorders, the present invention synthesized quercetin-based carbon dots (QA-CDs) with antibacterial and anti-inflammatory properties using quercetin (QU) and 4-aminophenol (4-AP) as raw materials via a simple one-step hydrothermal method. In vitro experimental results showed that QA-CDs exhibited significant antibacterial activity and ROS scavenging ability, and effectively regulated the expression levels of inflammatory-related factors. In vivo studies using clinically relevant models of peritonitis and pneumonia demonstrated that QA-CDs effectively exerted antibacterial activity while modulating the inflammatory microenvironment, thereby exhibiting significant dual functional properties. This unique property is of great significance as it not only establishes a new approach for the application of flavonoid-based compounds, but also provides a promising research framework for the development of innovative bioactive materials with dual therapeutic effects. This invention can greatly promote the development of innovative strategies in the field of bacterial infection treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a microscopic characterization image of the QA-CDs prepared in Example 1;

[0022] Figure 2 The test results of the antimicrobial activity of QA-CDs were evaluated by their inhibitory effects on the growth of Escherichia coli and Staphylococcus aureus;

[0023] Figure 3 The results of the study on the antibacterial mechanism of QA-CDs;

[0024] Figure 4 The biocompatibility analysis results of QA-CDs;

[0025] Figure 5 The results of the study on the antioxidant and anti-inflammatory functions of QA-CDs;

[0026] Figure 6 The results of the study on the antibacterial properties of QA-CDs against methicillin-resistant Staphylococcus aureus (MRSA) infection in mice animal model are presented. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0028] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Materials and reagents used in the following examples are commercially available unless otherwise specified. In the following examples, where specific conditions are not specified, the experiments were conducted under conventional conditions or those recommended by the manufacturer. Reagents and instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0030] Example 1

[0031] A quercetin-based carbon dot with antibacterial and anti-inflammatory properties, the preparation method of which comprises the following steps:

[0032] S1: 0.2 g of quercetin (QU) and 0.2 g of 4-aminophenol (4-AP) were added to 40 mL of pure water, mixed thoroughly, and then ultrasonicated for 10 minutes. The resulting mixed solution was transferred to a polytetrafluoroethylene-lined autoclave and heated in a thermostat at 200°C for 8 hours.

[0033] S2: After the reaction, the mixture was naturally cooled to room temperature, and the product was preliminarily filtered using filter paper and then finely filtered through a 0.22 μm membrane filter. Finally, the filtrate was freeze-dried to obtain a powdered product, namely quercetin-based carbon dots, denoted as QA-CDs, which was stored in a refrigerator at 4°C for further use.

[0034] Performance testing:

[0035] 1. Microscopic representation of QA-CD

[0036] Figure 1 QA-CDs were synthesized by a hydrothermal method using QU and 4-AP in a 1:1 ratio. Morphological analysis using transmission electron microscopy (TEM) showed that QA-CDs exhibited a classic spherical structure with good dispersion. The particle size distribution was found to be in the range of 2-6 nm, with an average diameter of 3.23 nm, which is consistent with the typical particle size definition of CDs. Fluorescence spectroscopy was used to study the optical properties of QA-CDs. It was found that the recorded fluorescence spectra varied with the change of excitation wavelength, ranging from 315 nm to 340 nm, and the optimal emission wavelength of 387 nm was obtained at an excitation wavelength of 330 nm. As shown in the figure, the UV absorption spectrum of QA-CDs showed two characteristic absorption peaks. The characteristic absorption peaks observed at 230 nm and 297 nm can be attributed to the aromatic sp 2The π-π* electronic transitions of the C=C bonds within the hybridized carbon domains indicate the presence of distinct aromatic conjugated structures within the material. This result confirms that the prepared QA-CDs retain some structural features of the precursor molecules QU and 4-AP. As shown in the figure, the zeta potential of the QA-CDs was measured to be -24.17 mV. Considering the negatively charged surfaces of most mammalian cells, it can be inferred that the material is unlikely to induce biotoxicity through electrostatic interactions. The elemental composition and chemical state of the QA-CDs were further analyzed using Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). As shown in the figure, the absorption bands observed at 1230, 1280, and 1470 cm⁻¹ are attributed to the stretching vibrations of the CN, COC, and C=C bonds, respectively. In addition, bands at 1610, 3280, and 3340 cm⁻¹ correspond to the stretching vibrations of the C=O, -OH, and NH groups. The FT-IR spectra of QA-CDs showed absorption bands at the same positions as those found in the spectra of quercetin (QU) and 4-aminophenol (4-AP), indicating that the functional group characteristics of the precursor were retained in the synthesized QA-CDs. XPS was used to further investigate the elemental composition and chemical state of QA-CDs. The high-resolution XPS full spectrum of QA-CDs showed that carbon (C) was the main element, accounting for 57.06%, followed by oxygen (O), accounting for 36.76%, while nitrogen (N) accounted for only 6.18%. These findings, along with the overall characterization results, confirmed the successful synthesis of QA-CDs while retaining the bioactive characteristics of QU and 4-AP. The presence of abundant functional groups on the surface further indicated that QA-CDs possess potential antibacterial and anti-inflammatory properties.

[0037] 2. In vitro antibacterial experiments of QA-CDs

[0038] Figure 2The antibacterial activity of QA-CDs was evaluated by evaluating their inhibitory effects on the growth of Escherichia coli and Staphylococcus aureus. Treatment with QA-CDs at varying concentrations significantly inhibited bacterial growth (105 CFU / mL). As shown in the figure, the inhibitory effect increased with increasing QA-CDs concentration. The minimum bactericidal concentrations (MBCs) for E. coli and S. aureus were 100 μg / mL and 25 μg / mL, respectively. To further evaluate the bactericidal efficiency of each concentration, colony counts were performed, as shown in the figure. The antibacterial activity of QA-CDs was observed to be concentration-dependent. At a concentration of 2.5 μg / mL, the inhibition rates against E. coli and S. aureus were 52.4% and 49.3%, respectively. As the concentration of QA-CDs increased, the antibacterial activity increased, ultimately achieving complete inhibition (100%) against E. coli and S. aureus at concentrations of 100 μg / mL and 25 μg / mL, respectively. To further evaluate the broad-spectrum antimicrobial efficacy of QA-CDs, the range of experimental bacterial strains was expanded to include Escherichia coli, Staphylococcus aureus, MRSA, Bacillus subtilis, and Pseudomonas aeruginosa. A comparative study was then conducted to evaluate the antimicrobial activity of QA-CDs against these bacteria compared with several commonly used antibiotics. The results shown in the figure demonstrate that the antimicrobial efficacy of QA-CDs is comparable to that of commonly used antibiotics. Specifically, QA-CDs exhibited the best antimicrobial activity against E. coli and Staphylococcus aureus. For MRSA and B. subtilis, the antimicrobial activity of QA-CDs was consistent with that of antibiotics, while the antimicrobial activity against Pseudomonas aeruginosa was superior to that of sulfadiazine but weaker than that of other antibiotics. These findings further demonstrate the potential of QA-CDs as broad-spectrum antimicrobial agents.

[0039] The antibacterial test steps of QA-CDs are as follows:

[0040] 1. Bacterial Culture

[0041] Prepare 1 liter of LB broth containing 10 g of tryptone, 5 g of yeast extract, and 0.5 g of NaCl for culturing Staphylococcus aureus, Escherichia coli, MRSA, Pseudomonas aeruginosa, and Bacillus subtilis. Incubate the culture at 37°C with shaking at 160 rpm for 12 hours.

[0042] 2. Co-culture of Bacteria and QA-CDs

[0043] Use Gram-negative (G - ) Escherichia coli and Gram-positive (G + ) Staphylococcus aureus was used as a model strain for antimicrobial evaluation. A microbial suspension in the logarithmic growth phase was prepared and diluted with LB broth to a final microbial concentration of 1×10 5CFU / mL. Subsequently, 180 μL of the diluted microbial suspension was added to a 96-well plate, followed by 20 μL of QA-CDs dissolved in PBS at final concentrations of 0, 2.5, 5, 10, 25, 50, 75, 100, 150, and 200 μg / mL. The 96-well plate was incubated at 37°C for 12 hours. Following incubation, the bacterial culture was serially diluted with PBS to the appropriate concentration, and 100 μL of each dilution was plated on LB agar plates. The plates were inverted and placed in a 37°C incubator for 24 hours.

[0044] 3. Comparison of antibacterial activity

[0045] Microbial suspensions in the logarithmic growth phase, including Staphylococcus aureus, Escherichia coli, MRSA, Pseudomonas aeruginosa, and Bacillus subtilis, were diluted in LB broth to a final concentration of 1 × 10 5 CFU / mL. Then, 180 μL of the suspension was pipetted into a 96-well plate, and 20 μL of chloramphenicol (CP), sulfadiazine (SD), ciprofloxacin (CH), and QA-CDs (QA) (100 μg / mL) were added to each bacterial culture. After incubation for 12 hours, the turbidity at 600 nm was measured using a microplate reader. Subsequently, the culture was serially diluted with PBS to an appropriate concentration, and 3 μL of each dilution was pipetted onto an LB agar plate. The plate was inverted and incubated at 37°C for 24 hours.

[0046] 4. Effects of QA-CDs on bacterial cell morphology

[0047] Escherichia coli and Staphylococcus aureus were treated with 20 μg / mL and 50 μg / mL of QA-CDs, respectively. After incubation at 37°C and 160 rpm for 7 hours, the effects of CDs on bacterial morphology were observed using scanning electron microscopy (SEM) and electron microscopy (TEM). SEM samples were prepared as follows: bacteria were centrifuged and washed with PBS to pellet the solution. The bacteria were then fixed in 2.5% glutaraldehyde for 3 hours. The fixed bacterial suspension was then centrifuged, washed with PBS, and dehydrated for 15 minutes using increasing concentrations of ethanol (30%, 50%, 70%, 90%, and 100%). The bacterial suspension was then resuspended in absolute ethanol, dropped onto a wafer, dried, and prepared for gold sputtering. A control group of bacteria, untreated with QA-CDs, was treated using the same procedures as the experimental groups and observed using SEM. TEM sample pretreatment was similar to that for SEM: bacteria were fixed in 2.5% glutaraldehyde and the fixative was then washed with PBS to remove the fixative. Bacteria samples were adsorbed onto carbon-coated copper grids, then covered with an appropriate amount of negative dye solution. After removing excess dye, the sample was dried and finally examined under TEM.

[0048] 5. Zeta potential measurement

[0049] Dilute the cultures of Escherichia coli and Staphylococcus aureus to 1 × 10 8 The concentration of CFU / mL was determined and the zeta potential of the bacterial solution was subsequently measured. The zeta potential of the QA-CDs stock solution and the bacterial suspension incubated with QA-CDs were also measured.

[0050] 6. Live / dead bacterial staining

[0051] Bacterial suspensions of Escherichia coli, Staphylococcus aureus, and MRSA were prepared at a concentration of 1 × 108 CFU / mL, with 1 mL of each suspension. A control and experimental group were established for each bacterial type. The experimental group received 100 μL of a 100 μg / mL QA-CDs solution, while the control group received the same volume of PBS. All samples were incubated at 37°C for 1 hour. The experimental group was incubated at 4000 rpm for 1 hour. Subsequently, the samples were centrifuged at 4000 rpm for 5 minutes, the supernatant discarded, and the samples were washed twice with PBS, each wash at 4000 rpm for 5 minutes. Finally, the bacterial pellet was resuspended in 1 mL of PBS. 3 μL of calcein-AM was added to each tube and incubated at room temperature in the dark for 20 minutes. After centrifugation, the supernatant was discarded, and the bacterial pellet was resuspended in 1 mL of PBS. 5 μL of propidium iodide (PI) was added and incubated at room temperature for 5 minutes. After centrifugation, discard the supernatant and resuspend the bacterial pellet in 1 mL of PBS. Pipet 3 μL of the resuspended solution onto a coverslip, allow to dry naturally in the dark, and photograph using a Nikon confocal microscope.

[0052] 7. Nucleic Acid Concentration Measurement

[0053] Escherichia coli and Escherichia coli at a concentration of 1×105 CFU / mL were incubated with 25 μg / mL and 100 μg / mL of QA-CDs, respectively. Incubation conditions were 37°C and 160 rpm. After 12 hours, the supernatant was centrifuged at 5000 rpm for 5 minutes and collected. The nucleic acid concentration in the supernatant was quantified using an ultra-micro UV-visible spectrophotometer. An equal volume of bacteria treated with PBS was used as a control.

[0054] 3. Study on the antibacterial mechanism of QA-CDs

[0055] In order to investigate the mechanism of QA-CDs on bacteria, live / dead bacterial fluorescence staining was performed. Figure 3As shown, the control group for all three bacterial strains exhibited primarily green fluorescence, exceeding 80% of the total population, indicating high viability. In contrast, bacteria treated with QA-CDs (100 μg / mL) exhibited primarily red fluorescence, exceeding 80% of the total population, indicating a significant decrease in bacterial viability. To further understand the morphological effects of QA-CDs on bacteria, SEM and TEM were employed. As shown in the figure, SEM analysis revealed significant deformation and damage to the bacterial cell membrane after QA-CDs treatment compared to the control group. TEM further confirmed this bacterial membrane disruption, observing diffusion at the edge of the E. coli cell membrane after QA-CDs treatment. Furthermore, no intact bacterial structures were observed in Staphylococcus aureus treated with QA-CDs, with scattered, spherical QA-CDs present, indicating complete bacterial elimination by the QA-CDs. Further analysis was conducted to examine changes in zeta potential and nucleic acid concentration. Treatment with QA-CDs significantly increased nucleic acid concentration and the absolute value of zeta potential in both bacterial strains. These results indicate that QA-CDs interact with bacteria, compromise their structural integrity and cause leakage of cellular contents, ultimately leading to a significant decrease in bacterial viability.

[0056] 4. Biocompatibility Analysis of QA-CDs

[0057] Figure 4 In the study, the hemolysis rate of QA-CDs at different concentrations (5-200 μg / mL) was first evaluated in vitro. As shown in the figure, with the increase of QA-CDs concentration, the hemolysis rate increased from 0.107% to 2.899%, which is still far below the 5% threshold set by the International Organization for Standardization (ISO) standard. At the cellular level, mouse macrophage cell line RAW 264.7 cells were incubated with different concentrations of QA-CDs for 24 hours, and the cell viability was maintained at approximately 100%. These results collectively indicate that QA-CDs have excellent biocompatibility and safety.

[0058] 5. Research on the Antioxidant and Anti-inflammatory Functions of QA-CDs

[0059] As shown in the figure, QA-CDs exhibited approximately 50% scavenging ability of DPPH free radicals at various concentrations (5, 25, 50, 75, 100, 150, and 200 μg / mL) in vitro. Furthermore, ROS levels in RAW264.7 cells incubated with QA-CDs were assessed using a 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. As can be seen, QA-CDs at a concentration of 25 μg / mL almost completely eliminated intracellular ROS. These findings highlight the significant free radical scavenging activity of QA-CDs, suggesting that they may exert anti-inflammatory effects through an antioxidant mechanism. QU has been shown to have significant anti-inflammatory effects, and QA-CDs have been shown to be able to scavenge reactive oxygen species (ROS) in vitro. Based on these findings, the present invention further investigated the inflammation-repairing potential of QA-CDs. In the current study, the cell model of inflammation was primarily induced by lipopolysaccharide (LPS). Although the effect of LPS concentration on the production of inflammatory mediators is still unclear, some studies have used different concentrations of LPS for stimulation, including 0.05 μg / mL, 0.1 μg / mL and 1 μg / mL. Therefore, in the present invention, RAW264.7 cells were induced with different concentrations of LPS, and the relative mRNA expression levels of inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), transforming growth factor β1 (TGF-β1) and anti-inflammatory cytokine interleukin-10 (IL-10) were detected. As shown in the figure, when RAW264.7 cells were stimulated with 5 μg / mL of LPS, the highest relative expression of TNF-α was observed. In addition, the expression levels of other key inflammatory mediators also changed significantly. Based on these observations, a concentration of 5 μg / mL of LPS was selected for subsequent experiments to induce an inflammatory model in RAW264.7 cells. After incubation of LPS-induced RAW264.7 cells with various concentrations of QA-CDs, the figure shows that 50 μg / mL of QA-CDs most significantly downregulated TNF-α expression, and therefore this concentration was used as the therapeutic concentration for subsequent experiments. The study found that QA-CDs significantly reduced the relative mRNA expression of LPS-induced inflammatory factors, and that co-incubation with QA-CDs significantly increased the relative mRNA expression of the anti-inflammatory factor IL-10. Given their potent antibacterial activity, these findings also suggest that QA-CDs may help repair the inflammatory microenvironment after bacterial infection.

[0060] The anti-inflammatory experimental steps of QA-CDs are as follows:

[0061] 1. Cell Culture

[0062] RAW264.7 cell culture medium was prepared using DMEM basal medium supplemented with 10% fetal bovine serum. Cells were maintained in a cell culture incubator at 37°C with 5% CO2. Subculture was performed when the cell density reached approximately 80%.

[0063] 2. Clear intracellular ROS

[0064] Intracellular ROS levels were measured using a reactive oxygen species (ROS) detection kit. RAW264.7 cells were seeded at a concentration of 1.5 × 105 cells / mL in confocal microplates, and 2 mL of cell suspension was added to each dish. After 24 hours of incubation, the supernatant was aspirated to establish control and experimental groups. For the experimental groups, 2 mL of QA-CDs diluted in complete culture medium was added to achieve final concentrations of 5, 25, and 100 μg / mL, respectively. For the control group, an equal volume of PBS was added. After 6 hours of co-incubation, the supernatant was discarded, and DCFH-DA was diluted at a ratio of 1:1000 in serum-free DMEM basal medium. Then, 1 mL of the diluted DCFH-DA solution was added to each dish and incubated for 30 minutes. Subsequently, the supernatant was discarded, the dishes were washed twice with PBS, and fresh PBS was added. The ROS scavenging effect was observed using a Nikon confocal microscope with 488 nm excitation light.

[0065] 3. LPS-induced RAW264.7 cells

[0066] RAW264.7 cells were cultured at 1.5 × 10 5 Cells were seeded into 6-well plates at a concentration of 1 μg / mL, and 3 mL of cell suspension was added to each well. Blank and LPS groups were established. After 12 hours of incubation, the supernatant was discarded. The culture medium in the blank group was replaced with fresh culture medium, while the LPS group was treated with different concentrations of LPS (final concentrations of 0.05, 0.1, 1, 5, and 25 μg / mL, respectively). The cells were then incubated for an additional 24 hours.

[0067] 4. RNA Extraction

[0068] Total RNA was extracted from cells using the Solarbio Animal Tissue / Cell Total RNA Extraction Kit, and the RNA concentration was determined using a micro-volume UV-visible spectrophotometer.

[0069] 5. RT-qPCR Detection

[0070] Reverse transcription of extracted total RNA was performed according to the manufacturer's instructions. Synthesized cDNA was diluted 1:1 and subsequently used in qPCR reactions according to the protocol outlined in the qPCR guide.

[0071] VI. Study on the Antibacterial Properties of QA-CDs in MRSA-Infected Mouse Models

[0072] In the present invention, two infection animal models were established using MRSA to evaluate the antibacterial properties of QA-CDs in vivo. 100 μL MRSA (10 8 Peritonitis and acute pneumonia models were established using a 4-hour circulation assay (100 μg / mL) of QA-CDs (QA-CDs) and a 5-hour QA-CDs (QA-CDs)-treated group. After 4 hours of circulation, mice were randomly assigned to either a control group or a QA-CDs group. The QA-CDs group received 100 μL of QA-CDs (100 μg / mL), while the control group received an equal volume of PBS. Peritoneal fluid and lavage fluid from all major organs of the mice were collected to evaluate the antibacterial effect of QA-CDs after 24 hours. As shown, bacterial counts in the peritoneal fluid and organ lavage fluid of the control group were significantly elevated, while no bacterial growth was detected in the QA-CDs group. Quantification of MRSA in the bacterial suspension by colony counts further confirmed the antibacterial activity of QA-CDs. To further investigate whether bacteria persist in major organs, organ homogenates were prepared and bacterial growth was assessed by plate culture. The results showed that no bacterial growth was observed in the lungs of the QA-CDs-treated pneumonia model. In the peritonitis model, bacterial growth in the control group was primarily detected in the spleen, lungs, and kidneys, suggesting that intraperitoneal injection primarily infects these three sites. However, in the QA-CDs group, no bacterial colonies were observed in the spleen, and bacterial loads in the lungs and kidneys were significantly reduced compared with those in the control group. These findings collectively demonstrate the significant in vivo antibacterial efficacy of QA-CDs.

[0073] In this study, novel carbon dots (QA-CDs) were successfully synthesized via a one-step hydrothermal method using quercetin (QU) and 4-aminophenol (4-AP) as precursors. Systematic characterization using TEM, XPS, and FTIR revealed the properties of QA-CDs. In vitro antibacterial evaluation of QA-CDs against Escherichia coli and Staphylococcus aureus was performed at minimum bactericidal concentrations (MBCs) of 100 μg / mL and 25 μg / mL, respectively. Comprehensive toxicity testing was performed to evaluate the biosafety of QA-CDs, and the results demonstrated excellent biocompatibility. Further studies revealed that QA-CDs possess significant anti-inflammatory properties. Specifically, they exhibited potent reactive oxygen species (ROS) scavenging activity. Furthermore, RT-qPCR analysis revealed a significant downregulation of mRNA expression levels of inflammatory cytokines (TNF-α, IL-6, IL-1β, iNOS, and TGF-β1), while upregulation of the anti-inflammatory cytokine IL-10 was accompanied by an increase in mRNA expression of inflammatory cytokines. To systematically evaluate the in vivo antibacterial efficacy of QA-CDs, clinically relevant bacterial infection models of peritonitis and pneumonia were established. After treatment with QA-CDs, the growth of methicillin-resistant Staphylococcus aureus (MRSA) was significantly inhibited in both models. Enzyme-linked immunosorbent assay (ELISA) analysis of tissue homogenates showed that the expression of TNF-α and IL-1β proteins in the QA-CDs-treated group was significantly downregulated. In vivo experiments confirmed that QA-CDs not only exhibited strong antibacterial activity, but also effectively inhibited the overexpression of inflammatory factors. These findings indicate that QA-CDs have excellent antibacterial properties both in vitro and in vivo, as well as significant inflammation regulation capabilities. The present invention provides new perspectives and methods for the prevention and treatment of inflammatory-related diseases caused by bacterial infections.

[0074] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for preparing quercetin-based carbon dots with antibacterial and anti-inflammatory properties, characterized in that: The following steps are involved: S1: quercetin and 4-aminophenol were added to pure water, mixed, and sonicated. The resulting mixed solution was transferred to a high-pressure reactor and reacted under heating; S2: After the reaction is completed, the mixture is cooled to room temperature, filtered, and the filtrate is freeze-dried to obtain quercetin-based carbon dots.

2. The method for preparing quercetin-based carbon dots with antibacterial and anti-inflammatory properties according to claim 1, wherein: The reaction temperature in step S1 is 180-220° C., and the reaction time is 4-16 hours.

3. The method for preparing quercetin-based carbon dots with antibacterial and anti-inflammatory properties according to claim 2, wherein: Step S1 is specifically as follows: 0.1-0.4 g of quercetin and 0.1-0.4 g of 4-aminophenol are added to 20-40 mL of pure water, mixed thoroughly, and then ultrasonically treated for 5-20 minutes. The resulting mixed solution is transferred to a polytetrafluoroethylene-lined autoclave and reacted at 180-220° C. for 4-16 hours.

4. The method for preparing quercetin-based carbon dots with antibacterial and anti-inflammatory properties according to claim 1, wherein: Step S2 is specifically as follows: after the reaction is completed, the mixture is naturally cooled to room temperature, the product is preliminarily filtered using filter paper, and then finely filtered through a 0.2-0.3 μm membrane filter. Finally, the filtrate is freeze-dried to obtain quercetin-based carbon dots.

5. The method for preparing quercetin-based carbon dots with antibacterial and anti-inflammatory properties according to claim 1, wherein: The following steps are involved: S1: 0.2 g of quercetin and 0.2 g of 4-aminophenol were added to 40 mL of pure water, mixed thoroughly, and then ultrasonicated for 10 minutes. The resulting mixed solution was transferred to a polytetrafluoroethylene-lined autoclave and reacted at 200 °C for 8 hours; S2: After the reaction is completed, the mixture is naturally cooled to room temperature, and the product is preliminarily filtered using filter paper, and then finely filtered through a 0.22 μm membrane filter. Finally, the filtrate is freeze-dried to obtain quercetin-based carbon dots, which are stored at 4°C for future use.

6. A quercetin-based carbon dot, characterized in that It is prepared by the method according to any one of claims 1 to 5.

7. Use of the quercetin-based carbon dots as claimed in claim 6 as an antibacterial agent.

8. Use of the quercetin-based carbon dots according to claim 6 in the preparation of an antibacterial composition or an antibacterial drug.

9. Use of the quercetin-based carbon dots according to claim 6 as an anti-inflammatory preparation.

10. Use of the quercetin-based carbon dots according to claim 6 in preparing an anti-inflammatory composition or an anti-inflammatory drug.

Images