Isatis root derived carbon dots as well as preparation method and application thereof

Abstract

The invention discloses indigowoad root derived carbon dots as well as a preparation method and application thereof, and belongs to the technical field of novel biomedical materials. The method comprises the following steps: generating a carbonized product by a one-step hydrothermal method, and separating and purifying to obtain the indigowoad root-derived carbon dots. The carbon dots do not contain metal elements, are non-toxic and high in biological safety, and show good free radical scavenging activity, certain superoxide dismutase-like activity and good biocompatibility no matter in vitro or in vivo. On the basis, the indigowoad root-derived carbon dots also show excellent anti-inflammatory activity, can down-regulate the inflammatory level of cells, relieve the paralysis symptom of AD nematode, relieve LPS-induced memory deterioration of mice and treat LPS-induced mouse Alzheimer's disease, and can be further applied to preparation of drugs for treating neurodegenerative diseases.

Description

Technical Field

[0001] The present invention belongs to the fields of medical technology and new biomedical material technology, and specifically relates to traditional Chinese medicine-derived carbon dots and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is an incurable neurodegenerative disease and the main form of dementia in the elderly. It is characterized by progressive cognitive impairment and cognitive decline, which gradually affect patients' daily activities. Clinical and experimental evidence in recent years have shown that damage to the brain by reactive oxygen species (ROS) is now becoming a key harmful factor driving the pathogenesis of AD. Oxidative stress (OS) irreversibly damages cellular biomolecules and disrupts neuronal function. More and more scientific evidence supports the therapeutic role of antioxidants in preventing the onset of AD pathology and delaying the progression of AD pathology. In recent years, the discovery of antioxidant nanozymes has brought great prospects for the treatment of Alzheimer's disease (AD). In order to effectively eliminate reactive oxygen species (ROS), various metal-based nanoparticles have been developed for the treatment of AD. The currently reported cerium dioxide nanoparticles use the successful strategy of scavenging ROS through Nrf-2 / HO -1 Signaling pathways inhibited oxidative stress in AD mice, achieving the therapeutic effect of alleviating AD (Hu, Yu et al. “Functionalized Cerium Dioxide Nanoparticles with Antioxidative Neuroprotection for Alzheimer's Disease.” International Journal of Nanomedicine (2023): s434873.). This demonstrates the potential role of metallo-nanozymes in preventing and treating related inflammatory neurodegenerative diseases. However, their unstable elimination activity and low biocompatibility have severely limited the practical application of metallo-nanozymes in the biomedical field.

[0003] In particular, carbon-based nanomaterials offer advantages such as high chemical stability, high catalytic activity, low toxicity, and biocompatibility compared to metal-based nanozymes, and are expected to play a potential role in the prevention and treatment of neurodegenerative diseases. Carbon dots (CDs), a novel carbon-based nanomaterial with a size less than 10 nm, have been widely used in the biomedical field due to their inherent nanozyme activity, small size, good biosafety, and ease of surface modification and functionalization. Traditional Chinese medicine (TCM) can be used to treat and prevent Alzheimer's disease (AD). However, effectively delivering them to the lesion site is a major challenge. Their application is also limited by their poor water solubility, low bioavailability, and potential toxicity. Furthermore, the blood-brain barrier (BBB) ​​is a major obstacle to effective TCM delivery, significantly reducing its efficacy. Summary of the Invention

[0004] Based on the above-mentioned prior art, the present invention proposes Radix Isatidis-derived carbon dots, their preparation method, and applications. Using traditional Chinese medicine as a precursor, the present invention hydrothermally prepares these traditional Chinese medicine-derived carbon dots, which are highly water-soluble, low-toxic, small-particle, and metal-free. These carbon dots exhibit stable chemical properties, high free radical scavenging efficiency, and excellent biocompatibility, making them suitable for the treatment and prevention of Alzheimer's disease.

[0005] To achieve the above technical objectives, the present invention uses Radix Isatidis as a precursor, generates a carbonized product through a one-step hydrothermal method, and then obtains the Radix Isatidis-derived carbon dots through separation and purification. Specifically, the present invention adopts the following technical solutions:

[0006] First, the present invention provides a method for preparing isatis root-derived carbon dots, which comprises the following steps:

[0007] (1) Grinding the Chinese medicine into powder using a wall-breaking machine to obtain Chinese medicine powder;

[0008] (2) Weighing Radix Isatidis powder, adding it to the solvent, and transferring it into a polytetrafluoroethylene-lined reactor, heating the reaction to obtain a yellow carbon dot solution;

[0009] (3) The yellow carbon dot solution is post-treated to obtain isatis root-derived carbon dots.

[0010] In the above-mentioned method for preparing carbon dots derived from Radix Isatidis, preferably, the heating reaction temperature in step (1) is 180-220°C, and the reaction time is 12-20 h, more preferably 200°C for 16 h.

[0011] In the above-mentioned method for preparing carbon dots derived from Radix Isatidis, preferably, in step (2), 10 g of Radix Isatidis powder is weighed and dissolved in 100-400 mL of double-distilled water.

[0012] In the above-mentioned preparation method of isatis root-derived carbon dots, preferably, the post-treatment in step (3) comprises filtering the yellow carbon dot solution with a 0.22 μm filter membrane; collecting the filtrate and dialyzing it for 36-72 h using a 500-1000 molecular weight dialysis membrane; and vacuum freeze-drying to obtain a solid powder.

[0013] The dialysis solution used in step (3) is double-distilled water, which is replaced every 12 hours until the external solution becomes clear. The dialysis time can be appropriately extended or shortened depending on the sample. A solid powder is obtained after vacuum freeze-drying.

[0014] As a more preferred preparation method, it may further comprise the following steps:

[0015] (1) Weigh the Radix Isatidis powder and add it to the solvent. Transfer the solution into a polytetrafluoroethylene-lined reactor, heat it to 200 °C, heat it for 16 h, and then cool it to room temperature to obtain a yellow carbon dot solution.

[0016] (2) The solution is post-treated to obtain isatis root-derived carbon dots. The post-treatment comprises filtering the yellow carbon dot solution with a 0.22 μm filter membrane. The filtrate is collected and dialyzed for 48 hours using a 500 molecular weight cutoff dialysis membrane. The dialyzed liquid is collected and freeze-dried under vacuum to obtain a solid powder.

[0017] Secondly, the Radix Isatidis-derived carbon dots obtained by the preparation method of the present invention can be named RI-CDs. They do not contain metal elements, have an average diameter of 1-5 nm, a lattice spacing of 0.20-0.30 nm, and have functional groups such as -OH, C=C, and COC on their surface.

[0018] Third, the Radix Isatidis-derived carbon dots described in the present invention can be used in the preparation of drugs for scavenging DPPH free radicals. They can also be used in the preparation of superoxide dismutase-like enzymes. They can also be used in the preparation of anti-inflammatory drugs. They can also be used in the preparation of drugs for treating or alleviating memory loss. They can also be used in the preparation of drugs for treating or preventing Alzheimer's disease. They can also be used in the preparation of drugs for treating or preventing neurodegenerative diseases.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The isatis root-derived carbon dots prepared in the present invention have a small average particle size, are rich in oxygen- and nitrogen-containing functional groups on the surface, have good water solubility, and can be widely used in the biomedical field.

[0021] (2) The Radix Isatidis-derived carbon dots prepared in this invention contain no metal elements, are non-toxic, and have high biosafety. They also exhibit good free radical scavenging activity and certain superoxide dismutase-like activity both in vitro and in vivo, as well as good biocompatibility. Based on these findings, the Radix Isatidis-derived carbon dots can be effectively used in the preparation of drugs for the treatment of Alzheimer's disease (AD).

[0022] (3) Compared with natural enzymes, the preparation and purification cost of Isatis root-derived carbon dots is low, their chemical properties are stable, and they can work efficiently in harsh environments (strong acid, strong alkali) and disease conditions, providing new ideas for the application of nanomaterials in anti-oxidative stress.

[0023] (4) The isatis root-derived carbon dots prepared in the present invention can treat Alzheimer's disease and alleviate memory loss and behavioral disorders in mice, and have good application prospects in the preparation of drugs for Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The DPPH radical (A) and ABTS+ radical (B) scavenging abilities of the Radix Isatidis-derived carbon dots obtained in Example 1 of the present invention;

[0025] Figure 2 Transmission electron microscopy morphology (A), particle size distribution histogram (B), Fourier transform infrared spectrum (C), and SOD enzyme activity (D) of the Radix Isatidis derived carbon dots obtained in Example 1 of the present invention.

[0026] Figure 3 This figure shows the biosafety evaluation of the Radix Isatidis-derived carbon dots in Example 3 of the present invention. Figure A shows the cytotoxicity of RI-CDs against human neuroblastoma cells (SH-SY5Y); Figure B shows the uptake and imaging of RI-CDs by human umbilical vein endothelial cells (HUVEC); Figure C shows the number of movements of nematodes within 30 seconds after one week of RI-CD feeding; Figure D shows the body length of nematodes after one week of RI-CD feeding; Figure E shows the hemolysis rate of RI-CDs; Figure F shows an image of the RI-CD hemolysis experiment; and Figure G shows an H&E staining of sections of major mouse organs 14 days after tail vein injection of RI-CDs.

[0027] Figure 4This is an evaluation of the anti-inflammatory ability of the Isatis root-derived carbon dots at the cellular level in Example 4 of the present invention. Figure A shows a fluorescence image of DCFDA-ROS detection in LPS-induced SH-SY5Y cells after RI-CDs intervention; Figure B shows the effect of RI-CDs intervention on the ROS content in LPS-induced SH-SY5Y cells; Figure C shows the effect of RI-CDs intervention on Aβ-induced SH-SY5Y cytotoxicity; Figures D and E show the effect of RI-CDs on the mRNA levels of inflammatory factors TNF-α and IL-1β in LPS-induced SH-SY5Y cells, respectively.

[0028] Figure 5 Figure 5 shows the anti-AD effect of the Isatis Radix-derived carbon dots in the AD model nematode. Figure A shows the alleviating effect of RI-CDs intervention on the CL4176 AD paralysis model, while Figures B, C, and D show the effects of RI-CDs intervention on the levels of MAD, GSH, and SOD in the somatic cells of the CL4176 AD paralyzed nematodes.

[0029] Figure 6 Figure 6 shows the effect of Isatis Radix-derived carbon dots on the alleviation of LPS-induced Alzheimer's disease in Example 6 of the present invention. Figure A shows the behavioral test path for mice in a water maze; Figure B shows the number of times each group of mice crossed the platform area within 60 seconds after the hidden platform was removed; Figure C shows the escape latency of each group of mice to find the platform area within 60 seconds after the hidden platform was removed; and Figure D shows the proportion of time each group of mice spent swimming in the target quadrant within 60 seconds after the hidden platform was removed.

[0030] Figure 7 Figure 7 shows the effect of Isatis Radix-derived carbon dots on the alleviation of LPS-induced Alzheimer's disease in Example 7 of the present invention. Figure A shows the behavioral path of the mouse water maze test; Figure B shows the number of times each group of mice crossed the platform area within 60 seconds after the hidden platform was removed; Figure C shows the escape latency of each group of mice to find the platform area within 60 seconds after the hidden platform was removed; and Figure D shows the proportion of time each group of mice spent swimming in the target quadrant within 60 seconds after the hidden platform was removed. DETAILED DESCRIPTION

[0031] The following embodiments are further descriptions of the present invention to illustrate the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0032] Example 1

[0033] The specific steps for preparing the Isatis Radix-derived carbon dots are as follows:

[0034] (1) Radix Isatidis-derived carbon dot nanozymes (RI-CDs): 10 g of Radix Isatidis powder was weighed and added to 100 mL of double-distilled water. The solution was added to a polytetrafluoroethylene-lined reactor and heated in an oven at 200 °C for 16 h to obtain a yellow carbon dot solution.

[0035] (2) Pour the yellow carbon dot solution into a beaker and filter it with a 0.22 μm filter membrane. Then dialyze it with a 500 molecular weight dialysis membrane for 48 h. Filter the dialyzed solution with a 0.22 μm filter membrane and pour it into a glass culture dish. Refrigerate it at -20 °C and freeze-dry it with a vacuum freeze dryer to obtain a solid powder.

[0036] (3) Ultrapure water was added to the solid powder prepared above, and the resulting solution after ultrasonic dissolution was the Radix Isatidis-derived Carbon Dots (RI-CDs) solution for subsequent applications.

[0037] The preparation of different concentrations of Radix Isatidis derived carbon dot solutions can be obtained by referring to the synthesis and post-treatment methods of RI-CDs in steps (1) to (3) above.

[0038] Example 2

[0039] The basic physicochemical properties of the carbon dot nanozyme prepared in Example 1 were measured.

[0040] (1) DPPH free radical scavenging activity

[0041] A 0.3 mM DPPH ethanol solution was prepared, and then 100 μL of an aqueous solution of isatis root-derived carbon dots (5, 10, 50, 100, 150, 200, 250, 300, 350 μg / mL) was mixed with 1 mL of DPPH solution. After incubation in the dark for 30 min, the absorbance of each solution was measured using a microplate reader, and the clearance rate was calculated. Figure 1 In the figure, A reflects the DPPH radical scavenging ability of the Isatis Radix-derived carbon dots in Example 1. It can be seen that after the Isatis Radix-derived carbon dots were added, the DPPH radicals in the system were scavenged to varying degrees.

[0042] (2) ABTS + Free radical scavenging activity

[0043] Dilute ABTS + The solution was prepared so that the absorbance at 734 nm was 1, and then 100 μL of aqueous solution of carbon dots derived from Radix Isatidis (5, 10, 50, 100, 150, 200, 250, 300, 350 μg / mL) was mixed with 1 mL of ABTS. +The solutions were mixed and incubated in the dark for 5 min. The absorbance of each solution was measured using a microplate reader and the clearance rate was calculated. Figure 1 Figure B reflects the ABTS of the Isatis Radix-derived carbon dots in Example 1. + Free radical scavenging ability. It can be seen that after the addition of isatis root-derived carbon dots, the ABTS in the system + Free radicals are scavenged to varying degrees.

[0044] (3) Characterization of morphology and surface functional groups

[0045] The morphology of the Radix Isatidis-derived carbon dots (RI-CDs) was characterized by a JEM-F200 transmission electron microscope. Fourier transform infrared (FT-IR) spectra were collected using a Bruker Hyperion 2000 spectrometer. Figure 2 A in the figure reflects the morphology of the carbon dot nanozyme derived from Radix Isatidis, and the RI-CDs are evenly distributed ( Figure 2 In Figure B), the average diameter is 1.7 ± 0.9 nm and the lattice spacing is 0.22 nm, corresponding to the (100) crystal plane of graphite. Figure 2 C in the figure is the Fourier transform infrared spectrum of RI-CDs. -1 OH / NH stretching vibration peak, 1706 cm -1 The peak of the asymmetric stretching vibration of carboxyl C=O is 1650 cm -1 C=C stretching zone, 1100cm -1 It is the COC stretching vibration peak.

[0046] (4) Superoxide dismutase (SOD) activity assay

[0047] The SOD activity of RI-CDs was determined using the Nanjing Jiancheng Total Superoxide Dismutase Activity Detection Kit (WST-8 method). Figure 2 Figure D reflects the superoxide dismutase (SOD) activity of RI-CDs. When the RI-CDs concentration reached 45µg / ml, the SOD inhibition rate reached 50%, resulting in a calculated SOD activity of approximately 111.1 U / mg.

[0048] Example 3

[0049] Biosafety evaluation of Isatis root-derived carbon dots:

[0050] Cell level: RI-CDs with a final concentration of 0-0.35 mg / mL in Example 1 were co-cultured with human neuroblastoma SH-SY5Y cells for 24 h, and the survival rates of cells in different treatment groups were determined by CCK-8 assay. Figure 3As shown in Figure A, when the concentration of RI-CDs was as high as 0.35 mg / mL, the survival rate of SH-SY5Y was still greater than 80%, showing good biocompatibility. RI-CDs with a final concentration of 0.3 mg / mL were co-cultured with human umbilical vein endothelial cells (HUVEC) for 2 h and the cells were observed using a laser confocal microscope. Figure 3 As shown in Figure B, after 2 hours of co-culture, RI-CDs can be taken up by HUVEC cells and emit bright blue light under the 405 nm channel, indicating that RI-CDs have the potential to cross the blood-brain barrier (BBB).

[0051] Nematode worms were used as experimental animals to investigate the effects of feeding RI-CDs on body length and movement frequency. The experimental groups were divided into the following groups: a control group was fed Escherichia coli OP50, while an experimental group was fed OP50 mixed with RI-CDs at 300 μg / mL. Synchronized L4 nematodes were transferred to corresponding NGM plates using a picker. At least three plates per group, with approximately 200 nematodes per plate, were incubated inverted at 20°C. This was designated day 0. Every other day or two, nematodes on each plate were transferred to a fresh NGM plate. On day 7, at least 10 live nematodes were randomly selected from each group using a picker and gently placed on a water agar plate. Body length was measured and recorded using LAZ EZ software, using microscopy. Experiments were repeated three times. Nematode body length reflects both growth status and growth rate. Figure 3 Figures C and D respectively counted the number of movements and body length of nematodes in each group. The results showed that there was no significant difference in body length and number of movements between the mixed-feed nematodes and the control group.

[0052] Animal testing: 1 ml of fresh C57BL / 6 mouse blood was diluted 50-fold with 0.9% NaCl solution. One ml of the above solution was transferred to a tube and the RI-CDs solution from Example 1 was added at different concentrations to achieve final concentrations of 5, 150, and 300 μg / mL, respectively. Red blood cells treated with 1% Triton X-100 served as a positive control. A negative control group was treated with 0.9% NaCl solution. Figure 3 As shown in Figures E and F, RI-CDs did not cause hemolysis, indicating that RI-CDs are biosafe.

[0053] After 15 days of continuous tail vein injection of RI-CDs in Example 1, H&E staining of major organ sections was as follows: Figure 3 As shown in G, no obvious pathological damage was observed compared with the control group, demonstrating the safety of RI-CDs.

[0054] Example 4

[0055] The cell experiment was divided into four groups: the control group consisted of normally cultured SH-SY5Y cells; the model group (LPS group) consisted of SH-SY5Y cells induced to produce inflammation by lipopolysaccharide; and the low- and high-concentration RI-CDs groups were added to the model group, with different concentrations of RI-CDs prepared in Example 1 added and co-cultured for 24 hours. A reactive oxygen species detection kit was used to determine the level of intracellular reactive oxygen species in the presence or absence of RI-CDs ( Figure 4 (A). Figure 4 Middle B, quantitative analysis using Image J showed that compared with the model group, the level of intracellular reactive oxygen species was significantly reduced after RI-CDs intervention. In addition, CCK8 kit was used to detect the effect of RI-CDs on Aβ 1-42 The mitigation effect of cytotoxicity caused by Figure 4 As shown in C, Aβ 1-42 Inducing cytotoxicity and causing cell apoptosis, while RI-CDs intervention can alleviate Aβ 1-42 Induced cytotoxicity. Fluorescence quantitative PCR was used to detect the relative expression of inflammatory markers (TNF-α and IL-1β) in cells. Figure 4 As shown in Figures D and E, the RI-CDs prepared in Example 2 can reduce the expression of TNF-α and IL-1β, and exhibit a good anti-inflammatory effect at the cellular level.

[0056] Example 5

[0057] The anti-AD effect of RI-CDs was verified using CL4176 AD-type Caenorhabditis elegans as an AD nematode model. CL4176 strain can induce human Aβ 1-42 It is expressed in muscle, resulting in a paralysis phenotype and is widely used as an AD model. After culturing the synchronized CL4176 strain on NGM plates without RI-CDs at 15°C for 72 hours, the strain was transferred to NGM plates with or without RI-CDs and cultured at 25°C for 48 hours. The paralysis of the nematodes in each group was observed and recorded. Each group had at least three plates, with 30 nematodes per plate. Figure 5 As shown in middle A, the CL4176 worm strain exhibited a temperature-sensitive paralysis phenotype at 25°C, and RI-CDs intervention alleviated the paralysis of the nematodes.

[0058] Follow the instructions of the MDA, GSH and SOD content detection kit. The amount of MDA reflects the degree of peroxidation in the body and indirectly reflects the degree of cell damage. Figure 5 As shown in Figure B, the MDA content in the model group was significantly increased, and the MDA content after RI-CDs intervention was significantly reduced compared with the model group, indicating that RI-CDs intervention can effectively alleviate the temperature-induced cell damage in nematodes. GSH and SOD participate in the antioxidant function of organisms, reflecting the antioxidant capacity of cells. Figure 5 As shown in Figures C and D, the levels of GSH and SOD in the nematodes in the model group were significantly reduced. After RI-CDs intervention, the levels of GSH and SOD were increased compared with the model group, indicating that RI-CDs intervention can improve the antioxidant capacity of cells in nematodes in the AD model.

[0059] Example 6

[0060] The RI-CDs described in Example 1 were used to treat lipopolysaccharide (LPS)-induced Alzheimer's disease in mice. C57BL / 6 mice were randomly divided into a control group, an LPS model group, a low-concentration RI-CDs group (LPS + CDs, 0.25 mg / kg), and a high-concentration RI-CDs group (LPS + CDs, 15 mg / kg). An Alzheimer's disease model was established by intraperitoneal injection of 0.25 mg / kg of LPS for 7 consecutive days, while the control group received an equal volume of normal saline. Drugs were administered via tail vein injection for 15 consecutive days after model establishment. On the 16th day, behavioral training was performed in the Moss water maze (MWM). Mice were trained in the MWM for 60 seconds to locate a hidden platform and remain on the platform for 30 seconds. Mice were placed in quadrants 1, 2, 3, and 4 daily for 5 consecutive days. Spatial learning scores, such as escape latency to find the hidden platform and swimming speed, were recorded. After 5 days of training, a final test was performed by removing the platform and allowing the mice to swim freely in the memory area for 60 s to find the platform area.

[0061] like Figure 6 As shown in Figure A, RI-CDs intervention alleviated the memory decline of AD mice. After modeling, the LPS group was still unable to quickly find the hidden platform area after five days of training. The RI-CDs intervention group had a significantly higher number of crossing the platform area after five days of training than the LPS group ( Figure 6 Middle B), the escape latency represents the time taken by mice to find the hidden platform. 15 days after the RI-CDs tail vein injection, the escape latency of mice to find the platform was significantly shorter than that of the LPS group ( Figure 6 Middle C), the proportion of time spent searching in the target quadrant where the platform is located was significantly higher than that of the LPS group ( Figure 6 Middle D) shows that mice tend to search for the hidden platform in the target quadrant.

[0062] Example 7

[0063] The RI-CDs in Example 1 were used to treat lipopolysaccharide (LPS)-induced Alzheimer's disease in mice. C57BL / 6 mice were randomly divided into a control group, an LPS model group, and a high-concentration RI-CDs group (LPS+CDs, 15 mg / kg). An Alzheimer's disease model was established by intraperitoneal injection of 0.25 mg / kg of LPS for 7 consecutive days, and the control group was given an equal amount of normal saline. The mice were gavage-administered for 21 consecutive days after modeling. On the 16th day, behavioral training of the Moss water maze (MWM) was performed, and the mice were trained in the MWM for 60 seconds to find the hidden platform and stayed on the platform for 30 seconds. The mice were placed in quadrants 1, 2, 3, and 4 every day and trained for 5 consecutive days. The spatial learning scores such as the escape latency and swimming speed for finding the hidden platform were recorded. After 5 days of training, a final test was performed by removing the platform, and the mice were allowed to swim freely in the memory area for 60 seconds to find the platform area. As Figure 7 As shown in Figure A, RI-CDs intervention alleviated the memory decline of AD mice. After modeling, the LPS group was still unable to quickly find the hidden platform area after five days of training. The RI-CDs intervention group had a significantly higher number of crossing the platform area after five days of training than the LPS group ( Figure 7 Middle B), the escape latency represents the time taken by mice to find the hidden platform. 15 days after the RI-CDs tail vein injection, the escape latency of mice to find the platform was significantly shorter than that of the LPS group ( Figure 7 Middle C), the proportion of time spent searching in the target quadrant where the platform is located was significantly higher than that of the LPS group ( Figure 7 Middle D) shows that mice tend to search for the hidden platform in the target quadrant.

[0064] In summary, the Radix Isatidis-derived carbon dots proposed in this study possess free radical scavenging activity and superoxide dismutase-like properties, demonstrating low toxicity and good biosafety in cells, nematodes, and animals. Furthermore, these Radix Isatidis-derived carbon dots exhibit excellent anti-inflammatory activity, downregulating cellular inflammation, alleviating paralysis symptoms in AD nematodes, alleviating LPS-induced memory loss in mice, and treating LPS-induced Alzheimer's disease in mice. They can also be used to prepare drugs for the treatment of neurodegenerative diseases.

[0065] It should be noted that the technical contents of the present invention described above are only for the purpose of explaining and illustrating the technical essence of the present invention to enable those skilled in the art to understand the technical essence of the present invention. Therefore, the technical contents described above are not intended to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be based on the claims. Those skilled in the art should be aware that any modifications, equivalent substitutions, and improvements based on the substantial spirit of the present invention shall fall within the substantial protection scope of the present invention.

Claims

1. A method for preparing isatis root-derived carbon dots, characterized in that: The steps include: (1) Grind the Radix Isatidis into powder using a wall-breaking machine to obtain Radix Isatidis powder; (2) Weighing Radix Isatidis powder, adding it to the solvent, and transferring it into a polytetrafluoroethylene-lined reactor, heating the reaction to obtain a yellow carbon dot solution; (3) The yellow carbon dot solution is post-treated to obtain isatis root-derived carbon dots.

2. The method for preparing carbon dots derived from Radix Isatidis according to claim 1, wherein: In step (1), the temperature of the heating reaction is 180-220° C., and the reaction time is 12-20 h. In step (2), 10 g of the isatis root powder is weighed and dissolved in 100-400 mL of double-distilled water.

3. The method for preparing carbon dots derived from Radix Isatidis according to claim 1, wherein: The post-treatment in step (3) includes filtering the yellow carbon dot solution with a 0.22 μm filter membrane; collecting the filtrate and dialyzing it for 36-72 h using a 500-1000 molecular weight dialysis membrane; and vacuum freeze-drying to obtain a solid powder.

4. The Radix Isatidis-derived carbon dots obtained by the preparation method according to any one of claims 1 to 3, characterized in that: The carbon dots do not contain any metal elements, have an average diameter of 1-5 nm, a lattice spacing of 0.20-0.30 nm, and have -OH, C=C, and COC functional groups on their surface.

5. Use of the Radix Isatidis derived carbon dots according to claim 4 in the preparation of a drug for scavenging DPPH free radicals.

6. Use of the Radix Isatidis derived carbon dots according to claim 4 in the preparation of superoxide dismutase-like enzymes.

7. Use of the Radix Isatidis derived carbon dots according to claim 4 in the preparation of anti-inflammatory drugs.

8. Use of the Radix Isatidis derived carbon dots according to claim 4 in the preparation of a drug for treating or alleviating memory loss.

9. Use of the Radix Isatidis derived carbon dots according to claim 4 in the preparation of a drug for treating or preventing Alzheimer's disease.

10. Use of the Radix Isatidis derived carbon dots according to claim 4 in the preparation of a drug for treating or preventing neurodegenerative diseases.

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