Preparation method and application of 5-methyl kaempferol carbon quantum dots
By preparing 5-methylkaempferol carbon quantum dots with a particle size of 2–10 nm, the problem of limited efficacy of existing treatments for acute lung injury was solved, achieving highly efficient antioxidant and anti-inflammatory effects and promoting lung injury repair.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-03-27
AI Technical Summary
Existing antioxidant therapies for acute lung injury have limited efficacy, poor targeting, or significant side effects, necessitating more effective treatment strategies.
5-Methylkaempferol carbon quantum dots were prepared by ultrasonic and hydrothermal methods, with a particle size of 2–10 nm, and were used to scavenge reactive oxygen species and inhibit inflammatory responses.
5-Methylkaempferol carbon quantum dots have highly efficient antioxidant and anti-inflammatory effects, significantly reduce oxidative damage to lung epithelial cells, promote the repair of acute lung injury, and have good biocompatibility.
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Figure CN121736746A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and more specifically, to a carbon quantum dot based on 5-methylkaempferol and its application in the preparation of a medicament for treating acute lung injury. Background Technology
[0002] Acute lung injury (ALI) is a severe lung disease caused by a variety of factors, including infection, trauma, and toxin exposure. Its main characteristics are damage to alveolar epithelial cells and capillary endothelial cells, leading to increased alveolar-capillary barrier permeability, diffuse pulmonary edema, and acute hypoxic respiratory failure. Oxidative stress and inflammatory responses play crucial roles in the occurrence and progression of ALI. Excessive accumulation of reactive oxygen species (ROS) triggers an inflammatory cascade, causing alveolar macrophages and epithelial cells to release pro-inflammatory factors (such as TNF-α, IL-1β, and IL-6), exacerbating neutrophil infiltration and further damaging lung tissue. Furthermore, ROS can also increase pulmonary vascular permeability by acting on vascular endothelial cells, exacerbating pulmonary edema and ultimately leading to impaired gas exchange. Treatment strategies for ALI include treatment of the underlying disease, respiratory support, and pharmacological intervention. In recent years, new treatment methods have been continuously explored in clinical practice. Some alleviate inflammatory responses by inhibiting inflammatory factors, while others improve hypoxia by enhancing alveolar oxygenation or reduce pulmonary edema by promoting pulmonary fluid clearance. Drugs used in these methods include glucocorticoids, N-acetylcysteine, theophylline, antioxidants, cytokine modulators, pulmonary surfactant, fluid ventilation, inhaled nitric oxide, and recombinant protein C. However, these treatments still suffer from limited efficacy, poor targeting, or significant side effects, requiring further in-depth research and large-scale clinical trials to verify their effectiveness and safety. Summary of the Invention
[0003] This invention addresses the shortcomings of existing antioxidant treatments for acute lung injury by providing a method for preparing 5-methylkaempferol carbon quantum dots. These 5-methylkaempferol carbon quantum dots can efficiently scavenge reactive oxygen species, reduce oxidative stress damage, and inhibit inflammatory responses.
[0004] This invention provides a method for preparing 5-methylkaempferol carbon quantum dots, specifically including the following steps:
[0005] S1. Disperse 5-methylkaempferol in water, and then add sodium hydroxide solution to it;
[0006] S2. Place the solution in an ultrasonic cleaner and sonicate for 30 minutes.
[0007] S3. Transfer the solution to an autoclave, then place the sealed autoclave in a muffle furnace for heating. After the reaction is complete, allow it to cool naturally to room temperature.
[0008] S4. Remove insoluble substances, then centrifuge to obtain a crude carbon quantum dot solution, then dialyze it. After dialysis, freeze-dry the solution to obtain the product 5-methylkaempferol carbon quantum dots.
[0009] Furthermore, in step S1, the mass ratio of 5-methylkaempferol to sodium hydroxide is 1:1-2:3.
[0010] Furthermore, the specific steps of step S3 are as follows: the solution is transferred to an autoclave, and then the sealed autoclave is placed in a muffle furnace for heating to 180°C for 7 hours. After the reaction is completed, it is naturally cooled to room temperature.
[0011] The present invention also provides 5-methylkaempferol carbon quantum dots prepared by the above preparation method.
[0012] Furthermore, the carbon quantum dots mentioned above contain 63.99% carbon, 31.98% oxygen, and 4.03% hydrogen.
[0013] Furthermore, the excitation wavelength of the aforementioned carbon quantum dots is 307 nm, and the emission wavelength is 414 nm.
[0014] Furthermore, the particle size of the aforementioned 5-methylkaempferol carbon quantum dots is 2–10 nm.
[0015] The present invention also provides the use of the above-mentioned 5-methylkaempferol carbon quantum dots in the preparation of antioxidant and anti-inflammatory drugs or drugs for treating acute lung injury.
[0016] The beneficial effects of this invention are as follows:
[0017] This invention provides a method for preparing 5-methylkaempferol carbon quantum dots, which has the following advantages: it adopts a green synthesis process, has high safety, and good biocompatibility.
[0018] The carbon quantum dots of 5-methylkaempferol act as highly efficient ROS scavengers, exhibiting significant antioxidant and anti-inflammatory effects. They can significantly reduce oxidative damage to lung epithelial cells and effectively promote lung injury repair in acute lung injury.
[0019] The carbon quantum dots of 5-methylkaempferol prepared in this invention have higher anti-inflammatory activity and better intracellular delivery efficiency than ordinary kaempferol, and have a better repair effect on acute lung injury. Attached Figure Description
[0020] Figure 1 , 2 This is a transmission electron micrograph based on 5-methylkaempferol carbon quantum dots, in which... Figure 1The mass ratio of 5-methylkaempferol to sodium hydroxide is 1:1. Figure 2 The mass ratio of 5-methylkaempferol to sodium hydroxide is 2:3.
[0021] Figure 3 The fluorescence emission spectra of 5-methylkaempferol carbon quantum dots under different reaction conditions;
[0022] Figure 4 The results show the effects of different concentrations of 5-methylkaempferol carbon quantum dots on an LPS-induced human lung epithelial cell inflammation model.
[0023] Figure 5 The results are cytotoxicity test results based on the effects of different concentrations of 5-methylkaempferol carbon quantum dots on LPS-induced human lung epithelial cell inflammation model;
[0024] Figure 6 The results show the effects of 5-methylkaempferol carbon quantum dots of different particle sizes on an LPS-induced human lung epithelial cell inflammation model.
[0025] Figure 7 and Figure 8 The results show the in vitro effects of 5-methylkaempferol and 5-methylkaempferol carbon quantum dots on an LPS-induced human lung epithelial cell inflammation model.
[0026] Figure 9 The fluorescence excitation and emission spectra of 5-methylkaempferol carbon quantum dots are shown.
[0027] Figure 10 Fourier transform infrared spectra based on 5-methylkaempferol carbon quantum dots;
[0028] Figure 11 X-ray diffraction analysis based on 5-methylkaempferol carbon quantum dots;
[0029] Figure 12 X-ray photoelectron spectroscopy analysis based on 5-methylkaempferol carbon quantum dots;
[0030] Figure 13 The results of cytotoxicity tests based on 5-methylkaempferol carbon quantum dots;
[0031] Figure 14 For cell viability assay based on 5-methylkaempferol carbon quantum dots;
[0032] Figure 15 For the detection of ROS index in lung epithelial cells based on carbon quantum dots of 5-methylkaempferol;
[0033] Figure 16 Quantitative analysis of ROS fluorescence intensity in lung epithelial cells based on 5-methylkaempferol carbon quantum dots;
[0034] Figure 17 The results of CAT activity assay in animal tissues based on 5-methylkaempferol carbon quantum dots;
[0035] Figure 18 The results of GSH-PX activity assay in animal tissues based on 5-methylkaempferol carbon quantum dots;
[0036] Figure 19 The results are based on the determination of MDA content in animal tissues using 5-methylkaempferol carbon quantum dots.
[0037] Figure 20 The results of SOD activity detection in animal tissues based on 5-methylkaempferol carbon quantum dots;
[0038] Figure 21 The results of LDH activity assay in animal tissues based on 5-methylkaempferol carbon quantum dots;
[0039] Figure 22 H&E staining results of lung sections from animal tissues based on 5-methylkaempferol carbon quantum dots Detailed Implementation
[0040] Example 1: Preparation and characterization of carbon quantum dots based on 5-methylkaempferol under different NaOH mass ratios
[0041] 1. Experimental Methods
[0042] To determine the optimal precursor ratio for the synthesis of 5-methylkaempferol carbon quantum dots (MF-CQDs) under alkaline conditions, systematic experiments were conducted using different mass ratios. 5-methylkaempferol was prepared with sodium hydroxide at four ratios: 1:1, 1:1.5, 1:2, and 2:3, dissolved in 50 mL of ultrapure water, and ultrasonically dispersed for 30 min. The mixture was then transferred to a 100 mL polytetrafluoroethylene-lined high-pressure reactor and reacted at a constant temperature (180°C) for 7 h. After cooling, the resulting reaction solution was purified by centrifugation and dialysis, and the supernatant was collected for characterization and performance evaluation.
[0043] 2. Experimental Results
[0044] from Figure 1 and Figure 2 Transmission electron microscopy (TEM) revealed that when the mass ratio was within the range of 1:1–2:3, the product particle size distribution was concentrated in the 1–10 nm range, exhibiting uniform particle size, regular morphology, and good dispersibility. MF-CQDs synthesized at this ratio possessed higher emission intensity and quantum yield, and their surfaces contained abundant oxygen-containing functional groups, which facilitated binding to biological targets. Preparation and characterization of carbon quantum dots based on 5-methylkaempferol were also discussed.
[0045] Example 2: Study on the Influence of Temperature and Time Parameters on the Hydrothermal Synthesis of 5-Methylkaempferol Carbon Quantum Dots
[0046] 1. Experimental Methods
[0047] To optimize the hydrothermal carbonization reaction conditions of 5-methylkaempferol carbon quantum dots (MF-CQDs), a systematic experiment was conducted to evaluate the product properties using different combinations of temperature and time. In the experiments, with a fixed precursor mass ratio of 1:1, the reaction solution was subjected to four temperature groups: 170°C, 180°C, 190°C, and 200°C, with reaction times set at 6 h, 7 h, and 8 h for each group. All reactions were carried out in a sealed autoclave and then allowed to cool naturally to room temperature after completion.
[0048] 2. Experimental Results
[0049] from Figure 3 It can be seen that the CQDs obtained by heating at 180°C for 7 h have the strongest fluorescence emission intensity and stability, which are significantly better than other combinations.
[0050] Example 3: Comparison of the therapeutic effects of different concentrations of 5-methylkaempferol carbon quantum dots on an acute lung injury model
[0051] 1. Experimental Methods
[0052] To determine the optimal therapeutic concentration and biosafety of MF-CQDs in an in vitro inflammation model, different dose gradients (12.5, 25, 50, 100, 200, and 400 μg / mL) were used to intervene in an LPS-induced human alveolar epithelial cell inflammation model. After 6 hours of stimulation with LPS (10 μg / mL), cells were treated with different concentrations of MF-CQDs for 24 hours. Cell supernatant was then collected to detect the expression of inflammatory factors such as IL-6 and TNF-α, and cell viability and morphological changes were assessed.
[0053] 2. Experimental Results
[0054] from Figure 4 , 5 It can be seen that MF-CQDs can effectively downregulate the expression of inflammatory factors and improve cell morphology within the concentration range of 12.5–400 μg / mL, while maintaining good cell viability. No obvious cytotoxicity was observed. Even at high concentrations (400 μg / mL), although the anti-inflammatory effect was not further enhanced, the cells still showed high survival rate and intact morphology, suggesting that MF-CQDs have good cell biocompatibility over a wide concentration range.
[0055] Example 4: Evaluation of the therapeutic effect of 5-methylkaempferol carbon quantum dots of different particle sizes in an acute lung injury model
[0056] 1. Experimental Methods
[0057] To evaluate the intervention potential of MF-CQDs of different particle sizes on inflammatory responses at the cellular level, this study prepared MF-CQDs samples with particle sizes ranging from 2 to 10 nm by adjusting the precursor concentration and hydrothermal reaction time. Under constant conditions, the initial concentration of 5-methylkaempferol and the reaction time were adjusted, and the particle size was measured using transmission electron microscopy (TEM), yielding carbon quantum dots with average particle sizes of 2.4 nm, 4.8 nm, 7.1 nm, and 9.6 nm. These MF-CQDs of different particle sizes were then applied to an LPS-induced human alveolar epithelial cell inflammation model. After 24 hours of treatment, the expression levels of inflammatory factors such as IL-6 and TNF-α in the cell culture supernatant were detected, and cell morphological changes were observed under a microscope.
[0058] 2. Experimental Results
[0059] from Figure 6 The results showed that MF-CQDs with a particle size between 2 and 10 nm significantly reduced the levels of inflammatory factors, maintained intact cell morphology, and significantly alleviated inflammatory damage. In contrast, CQDs with a particle size of 1 nm, although able to rapidly enter cells, exhibited weak intracellular aggregation, short duration of effect, and limited anti-inflammatory activity. CQDs with a particle size greater than 10 nm showed relatively low intervention effects due to decreased endocytosis efficiency and impaired cellular uptake. In summary, MF-CQDs with a particle size between 2 and 10 nm demonstrated the best anti-inflammatory capacity and cell compatibility in the cell model, suggesting that this particle size range may be the ideal size parameter for MF-CQDs in the intervention of lung diseases.
[0060] Example 5: Comparative Study of the Therapeutic Effects of Regular Kaempferol and Carbon Quantum Dot Formulations in an Acute Lung Injury Model
[0061] 1. Experimental Methods
[0062] To verify the enhancing effect of carbon quantum dot delivery system on kaempferol efficacy, an in vitro comparative experiment was designed: LPS-treated human lung epithelial cells were treated with the same concentrations of ordinary kaempferol solution (12.5, 25, 50 μg / mL) and MF-CQDs synthesized based on an equal amount of kaempferol precursor. The ordinary kaempferol solution was a free 5-methylkaempferol solution prepared using dimethyl sulfoxide (DMSO) as a solvent, diluted in complete culture medium, and served as a control. After 24 hours of culture, cell supernatant was collected, and the secretion levels of inflammatory factors TNF-α and IL-6 were detected. Cell morphology and apoptosis were also observed.
[0063] 2. Experimental Results
[0064] from Figure 7 , Figure 8The results showed that the MF-CQDs group was significantly superior to the ordinary kaempferol group in reducing the release of inflammatory factors, with more normal cell morphology and a significantly decreased apoptosis rate. The average reduction in inflammatory factors reached 42.3%. Furthermore, the MF-CQDs group exhibited stronger cytoplasmic distribution signals in intracellular fluorescence imaging, suggesting better intracellular delivery efficiency. These results demonstrate that MF-CQDs exhibit higher anti-inflammatory activity and drug bioavailability in cell models, validating its potential application as a kaempferol carrier in the treatment of lung diseases.
[0065] Example 6: Preparation and characterization of carbon quantum dots based on 5-methylkaempferol
[0066] 1. Experimental Methods
[0067] Weigh 0.5 g of 5-methylkaempferol into a 100 mL beaker and add 20 mL of deionized water containing 0.5 g of sodium hydroxide. Place the solution in an ultrasonic cleaner (frequency 40 kHz, power 100 W) at 25 °C for 30 min to promote dissolution and uniform dispersion. Transfer the solution to a 25 mL Teflon-lined stainless steel autoclave, then place the sealed autoclave in a muffle furnace and heat to 180 °C for 6 h until the reaction is complete. Allow the hydrothermal reactor to cool naturally to room temperature. Open the reactor and filter the mixture using a 0.22 µm filter membrane to remove insoluble substances. Then centrifuge (8000 rpm, 30 min) to obtain a crude carbon quantum dot solution. Transfer the solution to a dialysis bag with a molecular weight cutoff of 500 Da. Dialysis was performed using 1L of deionized water, with the water being changed three times at 3, 6, and 10 hours. After dialysis, the solution was freeze-dried for 24 hours to obtain the product 5-methylkaempferol carbon quantum dots.
[0068] After successfully preparing 5-methylkaempferol carbon quantum dots, their physicochemical properties were systematically characterized. Their optical properties were analyzed by fluorescence spectroscopy, and their chemical composition, crystal structure, and surface functional groups were determined using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS).
[0069] 2. Experimental Results
[0070] from Figure 9 It can be seen from the measured CQD cys The optimal excitation wavelength is 307 nm, and the optimal emission wavelength is 414 nm.
[0071] from Figure 10 The wavenumber is observed at 3136.46 cm⁻¹. -1 The absorption peak is due to the stretching vibration of CH, at 1675.10 cm⁻¹. -1The absorption peak is due to the stretching vibration of the carbonyl group (C=O), at 1584.12 cm⁻¹. -1 The absorption peak is due to the stretching vibration of C=C, at 1399.98 cm⁻¹. -1 The absorption peak is due to the stretching vibration of CO.
[0072] from Figure 11 The absence of characteristic peaks indicates that the carbon point has an amorphous structure and should be compared with the XRD pattern of the active pharmaceutical ingredient.
[0073] from Figure 12 The data shows that the carbon point contains 63.99% carbon, 31.98% nitrogen, and 4.03% oxygen.
[0074] Example 7: In vitro test
[0075] 1. Experimental Methods
[0076] Take lung epithelial cells in the logarithmic growth phase, at a dose of 1×10 6 Cells were seeded per well in six-well plates. After the cells adhered and stabilized, they were randomly divided into five groups, with three parallel wells in each group: control group, LPS group (10 μg / mL), LPS + 12.5 μg / mL K-CQDs, LPS + 25 μg / mL K-CQDs, and LPS + 50 μg / mL K-CQDs. All cells were cultured in an incubator at 37°C and 5% CO2.
[0077] To systematically evaluate the toxicity and oxidative stress levels of different concentrations of K-CQDs on lung epithelial cells, cell viability and ROS were simultaneously measured under the same experimental conditions. For toxicity assessment, after each group was cultured, CCK-8 working solution (10% of the culture medium volume per well) was added, and the cells were incubated for another 2 hours. The absorbance was then measured at 450 nm using a microplate reader to reflect changes in cell viability.
[0078] Subsequently, to observe intracellular reactive oxygen species (ROS) levels, cells in each group were treated with dihydroethidium (DHE) and Hoechst dye for fluorescent labeling. The cells were incubated for another 1 hour, with gentle inversion every 10 minutes to ensure adequate probe contact with the cells. After incubation, MERGE images were acquired, and finally, fluorescence images were observed and acquired using an inverted fluorescence microscope to analyze ROS generation and morphological changes in lung epithelial cells under different treatment conditions.
[0079] 2. Experimental Results
[0080] The level of ROS clearance in lung epithelial cells by 5-methylkaempferol-based carbon quantum dots can be analyzed through in vitro experiments using DHE and Hoechst fluorescence staining. Figure 13 , 14 As shown in 15 and 16, carbon quantum dots based on 5-methylkaempferol can significantly reduce the ROS level of lung epithelial cells.
[0081] Example 8: In vivo experiment
[0082] 1. Implementation Method
[0083] An acute lung injury mouse model was established. Male, 6-8 week old SPF-grade C57BL6 mice were selected and weighed. Mice were anesthetized with 5% chloral hydrate at a dose of 6 μl / g via intraperitoneal injection, and then fixed in a supine position on the operating table. Sterile LPS (10 mg / kg) was administered directly to the lungs via a nebulizer, while sterile PBS was used as a control group. Mice were returned to their cages for continued rearing, and their numbers, weights, anesthesia doses, and LPS doses were recorded. After 24 hours, the mice were sacrificed, and lung tissue was harvested and its volume recorded.
[0084] After homogenizing a portion of lung tissue, centrifuging was performed, and the supernatant was collected for colorimetric analysis. Superoxide dismutase (SOD) was analyzed using the xanthine oxidase method; catalase (CAT) was determined by the H₂O₂ decomposition rate; glutathione peroxidase (GSH-PX) was analyzed using the 5,5'-dithiobis(2-nitrobenzoic acid) (DNTB) colorimetric method; malondialdehyde (MDA) was analyzed using the thiobarbituric acid (TBA) colorimetric method; and lactate dehydrogenase (LDH) release was used to assess alveolar epithelial cell damage. Finally, absorbance was measured using an ELISA reader, and the activities or contents of relevant indicators were calculated to clarify the role of oxidative stress in the development and progression of ALI.
[0085] Frozen sections (5-8 μm thick) of lung tissue were prepared for ROS detection. ROS were labeled with a 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe, and incubation and detection were performed according to the kit instructions. Cell nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI), and nuclear morphology was observed under a microscope. Lung tissue sections were observed under a confocal microscope. The red fluorescence generated by DCFH-DA-labeled ROS was observed, and the blue fluorescence of the cell nuclei was displayed by DAPI staining. Finally, images from different fluorescence channels were merged for image analysis, thus visually demonstrating the location and distribution of ROS in lung tissue.
[0086] 2. Implementation Results
[0087] from Figure 17 , 18As shown in Figures 19, 20, 21, and 22, using an LPS-induced mouse model of acute lung injury and detecting the activity of oxidative stress-related enzymes, 5-methylkaempferol carbon quantum dots significantly increased the activities of SOD, CAT, and GSH-PX, enhanced the body's antioxidant defense capacity, and reduced MDA content and LDH release levels, thus alleviating lipid peroxidation damage and cell damage in lung tissue. Therefore, 5-methylkaempferol carbon quantum dots have a definite and significant antioxidant and cytoprotective effect on LPS-induced acute lung injury in mice.
Claims
1. A method for preparing 5-methylkaempferol carbon quantum dots, characterized in that: Includes the following steps: S1. Disperse 5-methylkaempferol in water, then add sodium hydroxide solution; S2. Place the solution obtained in S1 into an ultrasonic cleaner and sonicate for 30 minutes. 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 carbon quantum dot solution, then dialyze it. After dialysis, freeze-dry the solution to obtain the product 5-methylkaempferol carbon quantum dots.
2. The preparation method according to claim 1, characterized in that: In S1, the mass ratio of 5-methylkaempferol to sodium hydroxide is 1:1-2:
3.
3. The preparation method according to claim 2, characterized in that: The specific steps of S3 are as follows: the solution obtained in S2 is transferred to an autoclave, and then the sealed autoclave is placed in a muffle furnace for heating to 180°C for 7 hours. After the reaction is completed, it is naturally cooled to room temperature.
4. 5-Methylkaempferol carbon quantum dots prepared by any one of the preparation methods of claims 1-3.
5. The 5-methylkaempferol carbon quantum dots as described in claim 4, characterized in that: The carbon quantum dots contain 63.99% carbon, 31.98% oxygen, and 4.03% hydrogen.
6. The 5-methylkaempferol carbon quantum dots as described in claim 4, characterized in that: The carbon quantum dots have an excitation wavelength of 307 nm and an emission wavelength of 414 nm.
7. The 5-methylkaempferol carbon quantum dots as described in claim 4, characterized in that: The particle size of the 5-methylkaempferol carbon quantum dots is 2–10 nm.
8. The use of the 5-methylkaempferol carbon quantum dots according to any one of claims 4-7 in the preparation of antioxidant and anti-inflammatory drugs or drugs for treating acute lung injury.