Preparation method of arginine-modified apigenin carbon dots and application thereof

Abstract

The application provides application of arginine-modified apigenin carbon dots in preparation of antioxidant anti-inflammatory drugs or drugs for treating epilepsy and a preparation method of the arginine-modified apigenin carbon dots, wherein the prepared arginine-modified apigenin carbon dots have higher anti-inflammatory activity than common apigenin and arginine, have better intracellular delivery efficiency, have better repair effect on epilepsy, and have biological safety.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of arginine-modified apigenin carbon dots in the preparation of drugs for treating acute status epilepsy. Background Technology

[0002] Epilepsy is a chronic, non-communicable brain disorder characterized by recurrent seizures. Seizures manifest as transient, involuntary convulsions, either localized or generalized, often accompanied by loss of consciousness and incontinence. They can also present as transient impairments of consciousness, perception, motor function, sensation, emotion, or other cognitive functions, severely impacting the quality of life for patients and their families. Furthermore, epilepsy patients often have more physical problems and a higher proportion of mental health comorbidities, such as anxiety and depression. Compared to the general population, people with epilepsy have approximately a three times higher risk of premature death, particularly in low- and middle-income countries and rural areas.

[0003] Although multiple potential pathological mechanisms may be involved in the pathogenesis of epilepsy, the cause remains unknown in approximately 50% of patients worldwide. Current treatment primarily relies on medication, with about 70% of patients achieving seizure control with antiepileptic drugs. However, about one-third of patients develop drug resistance. For drug-resistant epilepsy, treatment options have expanded to include epilepsy surgery, neuromodulation techniques, and the ketogenic diet. In recent years, to address the issue of drug resistance, the field of epilepsy treatment has become increasingly diversified, encompassing neuromodulation, minimally invasive surgery, and precision medicine, but their effectiveness and safety still require further validation through large-scale clinical studies. Therefore, the development of novel antiepileptic drugs remains a significant clinical need. Summary of the Invention

[0004] This invention addresses the shortcomings of existing antioxidant methods for treating epilepsy by providing a method for preparing arginine-modified apigenin carbon dots, which can effectively alleviate acute epilepsy symptoms.

[0005] This invention provides the application of arginine-modified apigenin carbon dots in the preparation of antioxidant and anti-inflammatory drugs or drugs for treating epilepsy.

[0006] Furthermore, the surface of the arginine-modified apigenin carbon dots contains amino and oxygen-containing functional groups.

[0007] Furthermore, the particle size of the arginine-modified apigenin carbon dots is 5-10 nm.

[0008] This invention also provides a method for preparing the above-mentioned arginine-modified apigenin carbon dots, comprising the following steps: S1. Disperse apigenin in water, then add sodium hydroxide solution; S2. Place the solution obtained in S1 into an ultrasonic cleaner and ultrasonically clean it. S3. Transfer the solution obtained in S2 to an autoclave, then place the sealed autoclave in a muffle furnace for heating, and allow it to cool naturally to room temperature after the reaction is complete. S4. Remove insoluble substances, then centrifuge to obtain a crude product carbon quantum dot solution, then dialyze it. After dialysis, freeze-dry the solution to obtain the product apigenin carbon dots.

[0009] S5. Dissolve the apigenin carbon dots and L-arginine obtained in S4 in water, heat under vibration, and further dialyze the solution after reaction to remove residual L-arginine molecules. Freeze-dry the solution to obtain the product Arg-Api CDs.

[0010] Furthermore, in S5 above, the molar ratio of apigenin carbon dots to L-arginine is 1:(1.5-4).

[0011] Furthermore, in S5 above, the molar ratio of apigenin carbon dots to L-arginine is 1:4.

[0012] This invention provides arginine-modified apigenin carbon dots as used in the above applications, and arginine-modified apigenin carbon dots prepared by any of the above preparation methods all possessing any one or more of the following functions: (1) It has targeting properties against inflammatory macrophages; (2) It has antioxidant properties; (3) Anti-apoptosis; (4) Promotes intracellular NO production.

[0013] The beneficial effects of this invention are as follows: This invention provides a method for preparing arginine-modified apigenin carbon dots, which has the following advantages: it adopts a green synthesis process, has high safety, and good biocompatibility.

[0014] Arginine-modified apigenin carbon dots act as highly efficient ROS scavengers, exhibiting significant antioxidant and anti-inflammatory effects, and can significantly reduce epilepsy symptoms.

[0015] The arginine-modified apigenin carbon dots prepared in this invention exhibit higher anti-inflammatory activity and better intracellular delivery efficiency compared to ordinary apigenin and arginine, resulting in better repair effects on epilepsy and improved biosafety. Their surface is rich in amino and oxygen-containing functional groups, which facilitates ROS scavenging. Due to enhanced cellular uptake and synergistic antioxidant properties, they effectively protect cells from inflammatory damage. They possess significant antioxidant capacity at the cellular level, mitigating oxidative damage. They effectively deliver L-Arg and promote intracellular NO production, contributing to the inhibition of epilepsy. They also exhibit anti-apoptotic effects and possess antioxidant and NO-regulating functions. Attached Figure Description

[0016] Figure 1This is a particle size distribution diagram of apigenin carbon dots modified with arginine at different molar ratios in Example 1; Figure 2 This is a potential diagram of apigenin carbon dots modified with arginine at different molar ratios in Example 1; Figure 3 This is an evaluation of the ROS scavenging ability of apigenin carbon dots modified with arginine at different molar ratios in Example 1; Figure 4 This is a transmission electron micrograph of apigenin carbon dots modified with arginine in Example 2; Figure 5 The image shown is the FT-IR image of apigenin carbon dots modified with arginine in Example 2. Figure 6 XPS image of apigenin carbon dots modified with arginine in Example 2; Figure 7 The image shows the XRD pattern of apigenin carbon dots modified with arginine in Example 2. Figure 8 The results of the CCK8 assay for cytotoxicity based on arginine-modified apigenin carbon dots in Example 3 are shown. Figure 9 The results of cell viability and mortality fluorescence staining based on arginine-modified apigenin carbon dots in Example 3 are shown. Figure 10 The results of the cellular uptake assessment based on arginine-modified apigenin carbon dots in Example 4; Figure 11 The results of cell uptake assessment based on arginine-modified apigenin carbon dots in Example 4 (inflammation-inducing macrophages). Figure 12 The results of the assessment of cellular ROS scavenging ability based on arginine-modified apigenin carbon dots in Example 5; Figure 13 The results of the assessment of NO uptake capacity in cells based on arginine-modified apigenin carbon dots in Example 6; Figure 14 This is an evaluation of the anti-apoptotic ability of cells based on arginine-modified apigenin carbon dots in Example 7; Figure 15 The results of the evaluation of protein expression levels for ROS clearance and inflammatory response in model animals based on arginine-modified apigenin carbon dots in Example 8; Figure 16 This is an evaluation of the quantitative results of the effect of arginine-modified apigenin carbon dots on the expression of proteins that clear ROS and inflammatory responses in model animals in Example 8; Figure 17 This is the evaluation result of the effect of arginine-modified apigenin carbon dots on the expression mRNA level of ROS- and inflammation-related genes in model animals in Example 8; Figure 18 This is the safety assessment result of arginine-modified apigenin carbon dots on the heart, liver, spleen, lungs, kidneys and other organs of model animals in Example 9. Detailed Implementation

[0017] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to examples. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained in the art without creative effort should fall within the scope of protection of the present invention.

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0019] Example 1: Preparation and characterization of arginine-modified apigenin carbon dots (Arg-Api CDs) based on different molar ratios 1. Experimental Methods To determine the optimal precursor molar ratio for arginine-modified apigenin carbon dots (Arg-Api CDs), four molar ratios of apigenin to L-arginine (1:1.5, 1:2, 1:3, and 1:4) were systematically investigated. The specific procedures were as follows: apigenin solution was placed in an ultrasonic cleaner and sonicated for 30 min until completely dissolved. The mixture was then transferred to a 50 mL reactor and reacted at 180 °C for 6 h. After the reaction, the resulting mixture was centrifuged at 9000 rpm for 20 min, and the supernatant was collected. Subsequently, the product was purified for 48 h using a dialysis bag with a molecular weight cutoff of 2000 Da. Finally, the dialysis buffer was freeze-dried to obtain the target product after removing insoluble substances. The solution after dialysis was freeze-dried to obtain the apigenin carbon dots. The obtained apigenin carbon dots and L-arginine in different molar ratios were dissolved in water and heated for 15 min at medium power (594 W) in a household microwave oven at 45 °C with vibration (microwave frequency 2.45 GHz, total microwave output 900 W). The resulting solution was further dialyzed to remove residual L-arginine molecules. The solution was freeze-dried to obtain products of 1.5 CDs, 2.0 CDs, 3.0 CDs, and 4.0 CDs, respectively. The supernatant was collected for subsequent characterization and performance testing.

[0020] 2. Experimental Results like Figure 1As shown, particle size analysis using dynamic light scattering (DLS) revealed that as the molar ratio of apigenin to L-arginine increased from 1.5 to 4.0, the hydrated particle size of the synthesized carbon dots (1.5 CDs, 2.0 CDs, 3.0 CDs, and 4.0 CDs) gradually increased, indicating that the nanoparticle size could be controlled by adjusting the precursor ratio during the synthesis process. Figure 2 As shown, the Zeta potential of all samples is negative, indicating that the Arg-Api CDs surface is generally negatively charged. The results are as follows... Figure 3 As shown, the fluorescence intensity decreased with increasing L-arginine concentration in the synthesis of Arg-Api CDs, and 4.0 CDs exhibited the strongest scavenging ability. This indicates that Arg-Api CDs possess concentration-dependent ROS scavenging ability, which is related to their surface functional groups (such as amino and phenolic hydroxyl groups).

[0021] The surface contains abundant oxygen-containing functional groups, which facilitates binding with biological targets. Figure 1-3 Based on the analysis results, we ultimately selected a molar ratio of 4:1 for L-arginine.

[0022] Example 2: Preparation and characterization of apigenin carbon dots based on amino acid modification 1. Experimental Methods The apigenin solution was placed in an ultrasonic cleaner and sonicated for 30 min until completely dissolved. The mixture was then transferred to a 50 mL reactor and reacted at 180 °C for 6 h. After the reaction, the resulting mixture was centrifuged at 9000 rpm for 20 min, and the supernatant was collected. Subsequently, the product was purified for 48 h using a dialysis bag with a molecular weight cutoff of 2000 Da. Finally, the dialysate was freeze-dried to obtain the target product after removing insoluble substances. The solution after dialysis was freeze-dried to obtain apigenin carbon dots. The obtained apigenin carbon dots and L-arginine were dissolved in water and heated in a household microwave oven at 594 W for 15 min under vibration at 45 °C (the frequency of the household microwave oven is 2.45 GHz, and the total microwave output is 900 W). The reacted solution was further dialyzed to remove residual L-arginine molecules. The solution was freeze-dried to obtain the product Arg-Api CDs. The morphology, surface functional groups, crystal structure, and elemental composition of the product were systematically characterized using techniques such as transmission electron microscopy (TEM), Fourier transform infrared spectroscopy, X-ray diffraction, and X-ray photoelectron spectroscopy.

[0023] 2. Experimental Results from Figure 4 As can be seen, Arg-Api CDs are approximately spherical with a uniform size distribution and a particle size of about 5-10 nm.

[0024] from Figure 5As can be seen, FT-IR displays at 3400 cm⁻¹ -1 (OH / NH), 1600 cm -1 Characteristic peaks are present at (C=O) and other locations, suggesting the presence of L-Arg and apigenin structures.

[0025] from Figure 6 The full spectrum shows the presence of C, O, and N elements; the C 1s peak in the fine spectrum can be fitted as CC / C=C, CO / CN, and C=O; the O 1s and N 1s peaks correspond to hydroxyl / carboxyl and amino / guanidinyl groups, respectively. XPS further confirms that the surface is rich in amino and oxygen-containing functional groups, which is beneficial for the material to remove ROS.

[0026] from Figure 7 The XRD pattern shows broad diffraction peaks, confirming that the carbon dots have an amorphous structure.

[0027] Example 3: Evaluation of cytotoxicity of different concentrations of materials and active pharmaceutical ingredients 1. Experimental Methods CCK8 assay: Raw264.7 cells were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μg / mL in 96-well plates and cultured for 24 h. Subsequently, cells were treated with Arg-Api CDs at concentrations of 0, 12.25, 25, 50, 100, 200, and 400 μg / mL, and cultured for another 24 h. Finally, the cytotoxicity of Arg-Api CDs against the Raw264.7 cell line was assessed using a CCK-8 assay.

[0028] Cell viability staining: Raw264.7 cells were stained at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 1000 cells / well in 96-well plates and cultured for 24 h. Afterward, the cells were treated with LPS (5 μg / mL) for 24 h, followed by treatment with free L-arginine (50 μg / mL), apigenin (50 μg / mL), and Arg-Api CDs (50 μg / mL) for 24 h. The culture medium was then replaced with a solution containing PI (2 μg / mL) and Calcein-AM (1 μg / mL). After 30 min of culture, the cells were washed three times with PBS buffer, and intracellular fluorescence intensity was observed using a high-content imaging system.

[0029] 2. Experimental Results The results are as follows Figure 8 As shown, Raw264.7 macrophages maintained a survival rate of over 80% at concentrations up to 400 μg / mL, demonstrating good biocompatibility. This low toxicity provides a safety basis for its subsequent in vivo application.

[0030] The results are as follows Figure 9 As shown, after Raw267.4 macrophage injury, the Arg-Api CDs group showed an increase in live cells (green) and a decrease in dead cells (red), which was superior to the free drug group. This confirms that Arg-Api CDs can more effectively protect cells from inflammatory damage, which is related to enhanced cellular uptake and synergistic antioxidant activity.

[0031] Example 4: Evaluation of the targeted uptake capacity of in vitro materials 1. Experimental Methods To evaluate the targeting effect of Arg-Api CDs on inflammatory macrophages, we performed an in vitro cell uptake experiment. Raw264.7 cells were incubated at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μg / mL in 96-well plates and cultured for 24 h. These cells were then stimulated with LPS (5 μg / mL) for 24 h to induce macrophage inflammation. Raw264.7 cells were co-cultured with FITC@Arg-Api CDs (200 ng / mL) for 4 h, followed by washing the cells three times with PBS buffer and staining the nuclei with Hoechst 33342 dye for 30 min to observe nuclear structure.

[0032] 2. Experimental Results The results are as follows Figure 10 and Figure 11 As shown, FITC@Arg-Api CDs induce inflammation in macrophages in Raw264.7 cells. Figure 11 ) and uninduced macrophages ( Figure 10 Compared to other methods, it has a stronger uptake capacity. This confirms that Arg-Api CDs have excellent cellular uptake capacity and target inflammatory macrophages, which is beneficial for precise drug delivery.

[0033] Example 5: Comparative Study of the Therapeutic Effects of Common Apigenin, L-Arginine, and Carbon Dot Preparations in an Epilepsy Model 1. Experimental Methods Raw264.7 cells were spaced at 5 × 10⁶ cells per well. 3Raw264.7 cells were seeded at a density of 1000 cells / well in 96-well plates and cultured for 24 h. Afterward, the cells were treated with LPS (5 μg / mL) for 24 h, followed by another 24 h of LPS treatment to damage the cells. The antioxidant capacity of the material was assessed by detecting intracellular reactive oxygen species (ROS) levels using a ROS fluorescent probe (DCFH-DA, 5 μg / mL). LPS-stimulated Raw264.7 cells were co-cultured for 24 h with PBS buffer, free L-arginine (50 μg / mL), apigenin (50 μg / mL), and Arg-Api CDs (50 μg / mL). Cells were then stained sequentially with DCFH-DA and DAPI, followed by three washes with PBS buffer. Intracellular fluorescence intensity was observed using a high-content imaging system, and the intracellular nitric oxide content was semi-quantitatively analyzed using ImageJ software.

[0034] 2. Experimental Results The results are as follows Figure 12 As shown, Arg-Api CDs significantly reduced intracellular ROS levels in inflamed Raw264.7 macrophages. This indicates that they possess significant antioxidant capacity at the cellular level and can alleviate oxidative damage.

[0035] Example 6: In vitro NO release capacity test of Arg-Api CDs 1. Experimental Methods Raw264.7 macrophages were stored at 5 × 10⁶ cells per well. 3 Raw264.7 cells were seeded at a density of 1000 cells / well in 96-well plates and cultured for 24 h. After treatment with LPS (5 μg / mL) for 24 h to induce cell damage, the cells were then treated with free L-arginine (50 μg / mL), apigenin (50 μg / mL), and Arg-Api CDs (50 μg / mL). Subsequently, the cells were stained with a nitric oxide fluorescent probe (DAF-FM DA, 5 μg / mL) for 30 min, followed by Hoechst 33342 staining at 37°C for 10 min. After washing the cells three times with PBS buffer, intracellular fluorescence intensity was observed using a high-content imaging system, and the intracellular nitric oxide content was semi-quantitatively analyzed using ImageJ software.

[0036] 2. Experimental Results The results are as follows Figure 13 As shown, the NO fluorescence intensity in the Arg-Api CDs group was significantly higher than that in the free L-Arg group, especially in inflammatory macrophages. This confirms that Arg-Api CDs can effectively deliver L-Arg and promote intracellular NO production, which may help suppress epilepsy.

[0037] Example 7 Evaluation of the anti-apoptotic ability of the material 1. Experimental Methods Raw264.7 macrophages were stored at 5 × 10⁶ cells per well. 3 Raw264.7 macrophages were seeded at a density of [number] cells per well in 96-well plates and cultured for 24 h. Then, LPS-stimulated Raw264.7 macrophages were co-cultured for 24 h with PBS buffer, free L-arginine (50 μg / mL), apigenin (50 μg / mL), and Arg-Api CDs (50 μg / mL). Following this, the cells were treated with an Annexin V-FITC apoptosis detection kit for 30 min, and then washed three times with PBS buffer. Flow cytometry was used to detect apoptosis and perform semi-quantitative analysis.

[0038] 2. Experimental Results The results are as follows Figure 14 As shown, the proportion of apoptotic cells in the Arg-Api CDs group was significantly lower than that in the injury group. This further confirms its anti-apoptotic effect, which is related to its antioxidant and NO regulation functions.

[0039] Example 8: Treatment evaluation of Arg-Api CDs in a rat epilepsy model 1. Experimental Methods Adult male SD rats were randomly divided into a control group, a pilucarpine epilepsy model group, and an Arg-Api CDs treatment group, with 12 rats in each group. Following the lithium chloride-pilucarpine model, rats were first injected intraperitoneally with lithium chloride (127 mg / kg) and pilucarpine (35 mg / kg). Thirty minutes before the pilucarpine injection, the rats were injected with atropine (1 mg / kg) to block the peripheral effects of pilucarpine. Seizures in the rats were graded according to the lacine scale. Grade IV or V seizures were considered generalized torticollis and termed status epilepticus (SE). When a seizure lasted 60 minutes or longer, ignition was considered successful, and stimulation was immediately stopped. When a rat experienced a Class IV seizure for 60 minutes, diazepam (7.5 mg / kg) was used to terminate the seizure. The control group received the same dose of saline. After successful modeling, rats in the treatment group were injected with Arg-Api CDs solution (50 mg / kg) via the tail vein once a day for 7 consecutive days to comprehensively evaluate the therapeutic effect of Arg-Api CDs on the rat epilepsy model. Rats were sacrificed by cervical spine fracture and hippocampal tissue was isolated.

[0040] The above samples were analyzed by Western blotting and qPCR in equal numbers. First, total hippocampal protein was extracted using RIPA lysis buffer, and its concentration was determined and quantified using the BCA method before Western blotting. Equal volumes of protein samples were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with primary antibodies against RPL11, SOD1, PTGS2 (COX-2), and the internal control β-actin. After washing, the membranes were incubated at room temperature for 2 hours with HRP-labeled secondary antibody. Finally, the membranes were developed using ECL chemiluminescence, and the band grayscale was semi-quantitatively analyzed using ImageJ software. Simultaneously, total RNA was extracted using TRIzol reagent, and its concentration was determined using a nanodrop™ spectrophotometer. Four μg of RNA was then used to synthesize cDNA via reverse transcriptase. Subsequently, amplification was performed on the Roche LightCycler real-time quantitative PCR instrument, and the expression levels of related genes were detected using the SYBR Green method. Three independent experiments were conducted, with β-actin as an internal reference gene. Finally, the relative expression levels of the genes were calculated and analyzed using the 2–ΔΔCT method.

[0041] 2. Experimental Results Western blotting and qPCR results together showed that Arg-Api CDs exhibited significant neuroprotective effects in the epilepsy model. Figure 15 As shown, the Western blotting bands visually reflect the differential expression of SOD1, PTGS2, and RPL11 proteins in hippocampal tissue. Further grayscale quantitative analysis (...) Figure 16 .AC) and RT-qPCR gene expression analysis ( Figure 17 The results (.AC) consistently showed that, compared with the control group, the protein of Sod1, which clears ROS, was significantly higher in the hippocampus of the model group rats. Figure 16 A) and mRNA ( Figure 17 The expression of A was significantly suppressed; the protein of the pro-inflammatory factor Ptgs2 ( Figure 16 B) and mRNA ( Figure 17 B) Expression was significantly upregulated, suggesting oxidative stress and inflammatory damage in the hippocampus of the model animals. After Arg-Api CDs intervention, the expression of Sod1 at both the transcriptional and translational levels in the treatment group was significantly restored compared to the model group, while the expression of Ptgs2 was effectively inhibited at both the protein and gene levels. Interestingly, the expression of Rpl11 mRNA was significantly reduced in the model group, but showed a significant recovery in the treatment group ( Figure 17 C); however, the expression level of RPL11 protein decreased to some extent in both the model group and the treatment group, but the difference was not statistically significant. Figure 16C). The asynchrony between the expression trends of RPL11 protein and mRNA indicates that Arg-Api CDs affect the stability of the protein and accelerate its hydrolysis.

[0042] Example 9: Preliminary safety testing of Arg-Api CDs in rats 1. Experimental Methods Healthy adult male SD rats (weighing 20–22 g) were randomly divided into a PBS control group and an Arg-Api CDs treatment group. Each group was further divided into two subgroups: Day 1 (acute observation point) and Day 21 (subacute observation point), with n=6 rats in each subgroup. Mice in the treatment group were injected with Arg-Api CDs solution (50 mg / kg, consistent with the treatment dose in Example 8) via tail vein injection, while the control group was given an equal volume of sterile PBS solution. The administration regimen was the same as the treatment group: once daily for 7 consecutive days. Subsequent analyses were performed on Day 1 (last administration) and Day 21 (last administration).

[0043] Rats were euthanized at the corresponding time points, and the heart, liver, spleen, lungs, and kidneys were rapidly dissected and separated. After rinsing with pre-cooled sterile PBS to remove residual blood, the tissues were immediately fixed in 4% paraformaldehyde buffer for 24 h. After fixation, the tissues were sequentially dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin to prepare continuous paraffin sections with a thickness of 3 μm. After dewaxing and hydration, the sections were stained with hematoxylin and eosin (H&E): the cell nuclei were stained with hematoxylin for 5 min and rinsed with tap water until the cell nuclei turned blue; then the cytoplasm was stained with eosin for 2 min, dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. The histopathological morphology of each tissue was observed using an optical microscope (Olympus BX53), and representative images were acquired and analyzed.

[0044] 2. Experimental Results To assess the in vivo biosafety of Arg-Api CDs, we performed H&E staining and histopathological analysis on the major organs (heart, liver, spleen, lung, and kidney) of rats on Day 1 and Day 21 after administration. The results are shown in Figure 18.

[0045] At the Day 1 acute observation point, all organs in both the PBS control group and the Arg-Api CDs treatment group showed normal histological morphology: myocardial fibers were arranged regularly, cell nuclei were clear, and there was no inflammatory infiltration or necrosis; liver lobule structure was intact, hepatocyte cords were arranged in an orderly manner, and no fatty degeneration or cell swelling was observed; the white pulp and red pulp of the spleen were clearly demarcated, and lymphocytes were normally distributed; alveolar structure was intact, and there was no thickening or edema in the septa; renal tubular epithelial cells were arranged neatly, and glomeruli were normal in morphology. There were no significant pathological differences between the two groups.

[0046] At the subacute observation point on Day 21, the histological manifestations of all organs in both groups remained good, consistent with the results on Day 1, and no delayed toxic damage was observed. The organ structures of both the control and treatment groups were normal, with no chronic inflammation, fibrosis, atrophy, or proliferative lesions, suggesting that Arg-Api CDs did not induce significant histopathological changes under long-term in vivo exposure.

[0047] In summary, H&E staining results showed that no significant pathological damage was observed in the major organs of rats treated with Arg-Api CDs at either the acute or subacute time points, and the histological morphology was highly consistent with that of the PBS control group. This preliminarily confirms that Arg-Api CDs have good biocompatibility in rats, providing crucial safety support for its further in vivo therapeutic application.

Claims

1. The application of arginine-modified apigenin carbon dots in the preparation of antioxidant and anti-inflammatory drugs or drugs for treating epilepsy.

2. The application as described in claim 1, characterized in that: The surface of the arginine-modified apigenin carbon dots contains amino and oxygen-containing functional groups.

3. The application as described in claim 1, characterized in that: The particle size of the arginine-modified apigenin carbon dots is 5-10 nm.

4. A method for preparing arginine-modified apigenin carbon dots as described in any one of claims 1-3, characterized in that: Includes the following steps: S1. Disperse apigenin in water, then add sodium hydroxide solution; S2. Place the solution obtained in S1 into an ultrasonic cleaner and ultrasonically clean it. S3. Transfer the solution obtained in S2 to an autoclave, then place the sealed autoclave in a muffle furnace for heating, and allow it to cool naturally to room temperature after the reaction is complete. S4. Remove insoluble substances, then centrifuge to obtain a crude product carbon quantum dot solution, then dialyze it. After dialysis, freeze-dry the solution to obtain the product apigenin carbon dots. S5. Dissolve the apigenin carbon dots and L-arginine obtained in S4 in water, heat under vibration, and further dialyze the solution after reaction to remove residual L-arginine molecules. Freeze-dry the solution to obtain the product Arg-Api CDs.

5. The method as described in claim 4, characterized in that: In S5, the molar ratio of apigenin carbon dots to L-arginine is 1:(1.5-4).

6. The method as described in claim 5, characterized in that: In S5, the molar ratio of apigenin carbon dots to L-arginine is 1:

4.

7. The arginine-modified apigenin carbon dots used in claim 1 and the arginine-modified apigenin carbon dots prepared by any one of claims 4-6 all possess any one or more of the following functions: (1) It has targeting properties against inflammatory macrophages; (2) It has antioxidant properties; (3) Anti-apoptosis; (4) Promotes intracellular NO production.

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