Puerarin carbon dots as well as synthesis method and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to puerarin carbon dots as well as a synthesis method and application thereof. The puerarin carbon dots are synthesized by taking puerarin as a carbon source and m-phenylenediamine as a nitrogen source. The invention also provides a synthetic method of puerarin carbon dots, which comprises the following steps: dispersing puerarin in an inorganic solvent to obtain a solution A; dissolving m-phenylenediamine in an inorganic solvent to obtain a solution B; adding the solution B into the solution A, heating, reacting, cooling and filtering to obtain filtrate; and filtering the filtrate by using a filter membrane, dialyzing the filtrate, and freeze-drying to obtain the puerarin carbon dots. The invention further provides application of the puerarin carbon dots in preparation of a disease diagnosis and treatment integrated reagent. The invention solves the problems that the existing puerarin is poor in water solubility, low in bioavailability, weak in self-fluorescence and difficult to be used for biological imaging, and the existing puerarin is difficult to be directly used for a nano diagnosis and treatment system.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a puerarin carbon dot, its synthesis method, and its application. Background Technology

[0002] Puerarin (7,4′-dihydroxy-8-β-D-glucosyl isoflavone) is the main active ingredient isolated from kudzu root and has been shown to possess various pharmacological activities, including antioxidant, anti-inflammatory, anti-fibrotic, and cardiovascular protective effects. However, due to the rigidity of the puerarin molecule and the scarcity of ionizable groups, its water solubility is extremely low (≈0.1 mg / mL). -1 It has an oral absorption rate of <10%, a plasma half-life of only 15-30 minutes, and is easily cleared from the body by glucuronidation, resulting in a bioavailability of less than 5%. Furthermore, it lacks active targeting capabilities, severely limiting its clinical application in disease treatment.

[0003] To improve its drug-like properties, researchers both domestically and internationally have recently attempted to encapsulate puerarin using nanocarriers such as liposomes, polymer nanoparticles, and solid lipid nanoparticles, hoping to improve its solubility and in vivo pharmacokinetic behavior. While these methods have improved the dissolution rate and cycle time of puerarin to some extent, they still have the following shortcomings: ① The carrier has a single function, only playing a "solubilizing-sustained release" role, and does not have the ability to respond to the disease microenvironment (such as ROS, pH, enzymes), thus failing to achieve lesion-specific drug release; ② The carrier itself has no diagnostic function, making it impossible to monitor the distribution and release process of the drug in vivo in real time, and making it difficult to achieve "therapeutic integration"; ③ The preparation process often involves high temperature, high pressure, or organic solvents, which can easily lead to the cleavage of the glucose group at the C-8 position of puerarin or the oxidation of the 7,4′-phenolic hydroxyl group, resulting in structural damage and decreased activity; ④ The resulting nano-formulations generally have a large particle size (>200 nm) and a wide distribution (PDI>0.3), making them easily captured by the reticuloendothelial system, resulting in significant accumulation in the liver and spleen, and potentially high long-term toxicity.

[0004] Therefore, there is an urgent need to develop a novel puerarin delivery system that combines light conversion, ROS response, pharmacological activity, and nanoscale size to achieve precise diagnosis and simultaneous treatment of diseases.

[0005] In addition, although traditional carbon dots (CDs) have been widely studied due to their excellent fluorescence properties, high photostability and low toxicity, their function is limited. They can only be used as passive fluorescent contrast agents and do not have any pharmacological activity. Therefore, they cannot play a role in the treatment process and thus cannot achieve the integration of diagnosis and treatment (Theranostics).

[0006] On the other hand, while existing ROS scavenging materials (such as nitrogen oxides, thiols, polyphenolic small molecules, or inorganic nanozymes) can locally reduce reactive oxygen species levels, their functions are also limited and lack real-time signal output capabilities. They cannot simultaneously monitor and provide feedback on changes in ROS concentration and therapeutic efficacy during treatment, resulting in a disconnect between treatment and monitoring. This makes it difficult to meet the needs of precision medicine for real-time assessment and dynamic adjustment of treatment strategies. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a puerarin carbon dot, its synthesis method, and its application, in order to solve the problems of poor water solubility and low bioavailability of existing puerarin, making it difficult to use directly in nanomedicine systems. It can also solve the problem of traditional carbon dots having a single function, making it difficult to achieve "therapeutic integration", as well as the problem of existing ROS scavenging materials (such as nitrogen oxides, thiols, polyphenolic small molecules, or inorganic nanozymes) having a single function, lacking real-time signal output capability, and being unable to synchronously monitor and provide feedback on ROS concentration changes and efficacy during treatment, resulting in a disconnect between "treatment and monitoring".

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A puerarin carbon dot, wherein the puerarin carbon dot is synthesized using puerarin as a carbon source and m-phenylenediamine as a nitrogen source.

[0009] Puerarin carbon dots can also be called antioxidant polyphenol carbon dots.

[0010] Based on the aforementioned technical methods, by using puerarin as a carbon source, the phenolic hydroxyl groups, carboxyl groups, and conjugated π domains abundant on the surface of puerarin carbon dots form an integrated "shell-drug" complex with the isoflavone skeleton of puerarin through hydrogen bonding-π stacking synergistic effects. This allows the carbon dots to achieve a five-in-one principle of "solubilization-stabilization-long cycling-tracing-metabolic inhibition," systematically solving the bottlenecks of poor water solubility, easy degradation, and low bioavailability of puerarin. Furthermore, the carbon dots not only emit light but also possess the therapeutic functions of puerarin, laying a platform for its nanomedicine applications. This effectively solves the problems of poor water solubility and low bioavailability of existing puerarin, making it difficult to directly use in nanomedicine systems.

[0011] Among them, the phenolic hydroxyl groups, carboxyl groups, and conjugated π domains on the carbon dots of puerarin form an integrated "shell-drug" complex with the isoflavone skeleton of puerarin through the synergistic effect of hydrogen bonding and π stacking. Specifically, this includes: ① The phenolic hydroxyl groups form multiple hydrogen bonds with the 3′,4′-OH of the B ring of puerarin, instantly disrupting its planar lattice, blocking the hydrophobic stacking between molecules, and increasing the apparent water solubility by 2 to 3 orders of magnitude; ② The conjugated domains of the carbon dots form π-π stacking with the C2=C3 and carbonyl groups of puerarin, which not only act as a "molecular scaffold" to fix the drug at the <4nm scale, but also inhibit the E-rin of puerarin through photoinduced electron transfer. ③ Ring-opening hydrolysis improves chemical stability by >5 times; ④ Nanoscale size (<4nm) and negatively charged surface (ζ≈−15mV) endow EPR effect and renal clearance with dual channels, avoiding retention by the reticuloendothelial system and prolonging plasma half-life by 4~6 times; ⑤ The carbon dots themselves have fluorescence emitted at 460nm after excitation at 350nm, which can track in vivo distribution in real time and realize the integration of "carrier-diagnosis"; ⑥ As a natural antioxidant, the polyphenol structure scavenge ROS, reduce the expression of P-gp and CYP3A4 oxidative stress, synergistically inhibit efflux and first-pass metabolism, and increase oral bioavailability from 4.6% to 28%. In PBS solutions with a concentration of 50μg / mL, the antioxidant polyphenol carbon dots (puerarin carbon dots) showed an approximately 9.88-fold increase in hydroxyl radical scavenging ability, an approximately 6.52-fold increase in DPPH scavenging ability, and an approximately 1.19-fold increase in superoxide radical scavenging ability compared to puerarin.

[0012] Puerarin carbon dots retain the original functional groups (phenolic hydroxyl, phenyl, and ether groups) of puerarin, while also possessing excellent fluorescence properties. The phenolic hydroxyl group can play an antioxidant and anti-inflammatory role; the benzene ring structure has anti-inflammatory functions; and in the structure of puerarin, the benzene rings are connected by ether bonds, forming the unique isoflavone structure, which enhances the anti-inflammatory effect.

[0013] Preferably, the particle size of the puerarin carbon dots is 2~6 nm.

[0014] Preferably, the particle size of the puerarin carbon dots is 2 nm.

[0015] By constructing ultra-small nanoparticles with a particle size of approximately 2 nm and uniform distribution, the biocompatibility of puerarin carbon dots was significantly enhanced, accelerating rapid renal clearance and minimizing long-term toxicity.

[0016] The present invention also provides a method for synthesizing puerarin carbon dots as described herein, comprising the following steps: Puerarin was dispersed in an inorganic solvent to obtain solution A; Dissolve m-phenylenediamine in an inorganic solvent to obtain solution B; Solution B is added to solution A, the mixture is heated to react, cooled, and filtered to obtain the filtrate. The filtrate was filtered through a filter membrane, then dialyzed, and freeze-dried to obtain puerarin carbon dots, i.e., antioxidant polyphenol carbon dots.

[0017] By utilizing the temperature difference-driven self-assembly of the "m-phenylenediamine-puerarin" binary system in an inorganic solvent: during the heating stage, m-phenylenediamine pre-organizes with the puerarin isoflavone skeleton through π-π stacking to form a homogeneous molecular complex; during the cooling stage, m-phenylenediamine undergoes mild oxidation-polymerization in a weakly acidic medium, generating a polyphenol carbon dot shell rich in phenolic hydroxyl groups in situ, while the hydrogen bond network tightly embeds puerarin in a core-shell structure of <4nm. The entire process requires no strong alkali, metal catalysts, or high-energy ultrasound, avoiding the oxidative breakage of the C-4′ hydroxyl group and C-ring double bond of puerarin, thus preserving its antioxidant and NF-κB inhibitory activities. The low-temperature nucleation and slow growth mechanism results in a particle size distribution of <10nm and a PDI <0.15, allowing direct glomerular filtration and achieving "zero residue" renal clearance. The m-phenylenediamine polymer shell has a natural phenolic network, endowing the nanomedicine with excellent water solubility and ROS scavenging ability, synergistically reducing the toxicity of the carrier itself, and providing a green and scalable universal platform for the high-activity and low-toxicity nanomedicine applications.

[0018] Preferably, the heating reaction is carried out at a temperature of 170~200℃ for 6~12 hours.

[0019] Preferably, the heating reaction temperature is 180°C and the heating reaction time is 8 hours.

[0020] Preferably, the molar ratio of puerarin to m-phenylenediamine is 1:2.

[0021] Preferably, the inorganic solvent is selected from water.

[0022] Preferably, the filtrate is filtered using a 0.22 μm filter membrane.

[0023] Preferably, the filtrate is dialyzed using an MV: 500 dialysis membrane for 70-74 hours.

[0024] Preferably, the step of dispersing puerarin in an inorganic solvent to obtain solution A specifically includes: adding puerarin to water, sonicating, and stirring for 30 minutes to obtain solution A.

[0025] Preferably, the step of dissolving m-phenylenediamine in an inorganic solvent to obtain solution B specifically includes: adding m-phenylenediamine to water and stirring to dissolve for 30 minutes to obtain solution B.

[0026] Preferably, the solution B is added to the solution A, heated to react, cooled, and filtered to obtain a filtrate. Specifically, the process includes: adding the solution B dropwise to the solution A, stirring for 2 hours, then transferring it to a hydrothermal reactor, reacting at 180°C for 8 hours, cooling, and then vacuum filtering three times to obtain the filtrate.

[0027] Preferably, the filtrate is filtered using a filter membrane, then dialyzed, and freeze-dried to obtain puerarin carbon dots. Specifically, the filtrate is filtered using a 0.22 μm filter membrane, then dialyzed using an MV: 500 dialysis membrane for 72 h, freeze-dried, and stored under light-protected conditions for later use.

[0028] Preferably, the prepared puerarin carbon dots emit fluorescence at a wavelength of 460 nm when irradiated with light at a wavelength of 350 nm.

[0029] Preferably, the prepared puerarin carbon dots emit fluorescence at a wavelength of 460 nm when irradiated with light at a wavelength of 645 nm.

[0030] Experimental verification has shown that the puerarin carbon dots prepared by the method of this invention not only possess the ability to excite 460nm fluorescence at 350nm, but also have excellent upconversion capabilities, allowing them to be excited at 460nm fluorescence using near-infrared light at 645nm with stronger penetrating power. This effectively solves the problem of traditional carbon dots having a single function, making it difficult to achieve "therapeutic integration." It also addresses the issue of existing ROS scavenging materials (such as nitrogen oxides, thiols, polyphenolic small molecules, or inorganic nanozymes) having a single function and lacking real-time signal output capabilities, thus failing to synchronously monitor and provide feedback on ROS concentration changes and therapeutic effects during treatment, leading to a disconnect between treatment and monitoring.

[0031] Preferably, the prepared puerarin carbon dots have ROS scavenging and fluorescence feedback functions.

[0032] The puerarin carbon dots prepared by the method of this invention utilize the correlation between light intensity and ROS concentration to achieve real-time fluorescence feedback on the ROS scavenging effect.

[0033] This invention also provides the application of puerarin carbon dots prepared by the synthesis method described herein in the preparation of integrated reagents for disease diagnosis and treatment.

[0034] Preferably, the disease includes at least one of osteoarthritis, acute lung injury, atopic dermatitis, allergic dermatitis, acne, and urticaria.

[0035] The beneficial effects of this invention are: The puerarin carbon dots of this invention, by using puerarin as a carbon source, enable the phenolic hydroxyl groups, carboxyl groups, and conjugated π domains on the surface of the polyphenol carbon dots to form an integrated "shell-drug" complex with the isoflavone skeleton of puerarin through the synergistic effect of hydrogen bonding-π stacking. Thus, the carbon dots, through the five-in-one principle of "solubilization-stabilization-long-cycle-tracing-metabolic inhibition", not only systematically solve the bottlenecks of poor water solubility, easy degradation, and low bioavailability of puerarin, but also enable the carbon dots to not only emit light, but also possess the therapeutic function of puerarin, laying the foundation for its nano-diagnostic and therapeutic applications.

[0036] The method for synthesizing puerarin carbon dots of the present invention utilizes the temperature difference-driven self-assembly of the "m-phenylenediamine-puerarin" binary system in an inorganic solvent: during the heating stage, m-phenylenediamine pre-organizes with the puerarin isoflavone skeleton through π-π stacking to form a homogeneous molecular complex; during the cooling stage, m-phenylenediamine undergoes mild oxidation-polymerization in a weakly acidic medium to generate a polyphenol carbon dot shell rich in phenolic hydroxyl groups in situ, while the hydrogen bond network tightly embeds puerarin in a <5nm core-shell structure. The entire process requires no strong alkali, metal catalysts, or high-energy ultrasound, avoiding the oxidative breakage of the C-4′ hydroxyl group and C-ring double bond of puerarin, thus preserving its antioxidant and NF-κB inhibitory activities. The low-temperature nucleation and slow growth mechanism results in a particle size distribution of <10nm and a PDI <0.15, allowing direct glomerular filtration and achieving "zero residue" renal clearance. The m-phenylenediamine polymer shell possesses a natural phenolic network, endowing the nanomedicine with excellent water solubility and ROS scavenging ability, synergistically reducing the toxicity of the carrier itself. This provides a green, scalable, and universal platform for the high-activity, low-toxicity nanomedicine applications, and has significant potential for widespread application in the field of pharmaceutical technology. Attached Figure Description

[0037] Figure 1 Images of Pue-CDs solutions under different illumination conditions; Figure 2 The results of fluorescence intensity excitation of Pue-CDs solution by irradiation at different wavelengths are shown in the figure. Figure 3 Infrared spectra of Puerarin and Pue-CDs; Figure 4 The fluorescence emission spectrum of Pue-CDs; Figure 5 Particle size distribution of Pue-CDs; Figure 6 Transmission electron microscope image of Pue-CDs; Figure 7 A comparison of the ·OH scavenging capabilities at different concentrations of Puerarin and Pue-CDs; Figure 8 O2 for different concentrations of Puerarin and Pue-CDs -Comparison chart of clearance capabilities; Figure 9 A comparison of DPPH scavenging capacity results at different concentrations of Puerarin and Pue-CDs; Figure 10 Image showing the results of cell viability and cell death staining stimulated by different concentrations of Pue-CDs; Figure 11 The image shows the results of the CCK-8 cell viability / toxicity colorimetric assay. Figure 12 The graph shows the linear relationship between the intensity of 460nm emission light generated by Pue-CDs under 360nm excitation light and the fluorescence intensity of ROS. Figure 13 Figure 1 shows the ROS fluorescence intensity of ATDC-5 after intervention with different Pue-CDs concentrations for IL-1β stimulation. Figure 14 Image showing the results of NF-κb immunofluorescence staining; Figure 15 The graph shows the ROS detection results of ATDC-5 after Pue-CDs intervention and IL-1β stimulation. Detailed Implementation

[0038] The following description, with reference to preferred embodiments, illustrates the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are merely illustrative of the present invention and not intended to limit the scope of protection of the present invention.

[0039] Example 1 A method for synthesizing puerarin carbon dots includes the following steps: S1. Add 2.5 mmol of puerarin to 30 mL of water, sonicate, stir and disperse for 30 min to obtain solution A; S2. Dissolve 1.2 mmol of m-phenylenediamine in 30 mL of water and stir for 30 min to obtain solution B; S3. Add solution B obtained in S2 dropwise to solution A obtained in S1, stir for 2 hours, then transfer to a 100 mL hydrothermal reactor, place in an oven at 180 °C and react for 8 hours, cool down, and then filter under vacuum three times to obtain the filtrate. S4. Filter the filtrate obtained in S3 using a 0.22μm filter membrane, then dialyze it using an MV: 500 dialysis membrane for 72 hours, freeze-dry it to form a powder, collect it, and obtain puerarin carbon dots, i.e., Pue-CDs. Store it under light-protected conditions for later use. When using it, simply weigh the powdered polyphenol carbon dots to prepare a solution of the required concentration.

[0040] Example 2 A method for synthesizing puerarin carbon dots includes the following steps: S1. Add 2 mmol of puerarin to 30 mL of water, sonicate, stir and disperse for 30 min to obtain solution A; S2. Dissolve 1 mmol of m-phenylenediamine in 30 mL of water and stir for 30 min to obtain solution B; S3. Add solution B obtained in S2 dropwise to solution A obtained in S1, stir for 2 hours, then transfer to a 100 mL hydrothermal reactor, place in an oven at 180 °C and react for 8 hours, cool down, and then filter under vacuum three times to obtain the filtrate. S4. The filtrate obtained in S3 was filtered through a 0.22 μm filter membrane, then dialyzed through an MV: 500 dialysis membrane for 72 h, freeze-dried to form a powder, collected, and puerarin carbon dots, i.e., Pue-CDs, were obtained and stored under light-protected conditions for later use.

[0041] Example 3 A method for synthesizing puerarin carbon dots includes the following steps: S1. Add 2.5 mmol of puerarin to 30 mL of water, sonicate, and stir to disperse for 30 min to obtain solution A; S2. Dissolve 1.2 mmol of m-phenylenediamine in 30 mL of water and stir for 30 min to obtain solution B; S3. Add solution B obtained in S2 dropwise to solution A obtained in S1, stir for 2 hours, then transfer to a 100 mL hydrothermal reactor, place in an oven at 170 °C and react for 8 hours, cool down, and then filter under vacuum three times to obtain the filtrate. S4. The filtrate obtained in S3 was filtered through a 0.22 μm filter membrane, then dialyzed through an MV: 500 dialysis membrane for 72 h, freeze-dried to form a powder, collected, and puerarin carbon dots, i.e., Pue-CDs, were obtained and stored under light-protected conditions for later use.

[0042] Example 4 A method for synthesizing puerarin carbon dots includes the following steps: S1. Add 2.5 mmol of puerarin to 30 mL of water, sonicate, and stir to disperse for 30 min to obtain solution A; S2. Dissolve 1.2 mmol of m-phenylenediamine in 30 mL of water and stir for 30 min to obtain solution B; S3. Add solution B obtained in S2 dropwise to solution A obtained in S1, stir for 2 hours, then transfer to a 100 mL hydrothermal reactor, place in an oven at 200 °C and react for 8 hours, cool down, and then filter under vacuum three times to obtain the filtrate. S4. The filtrate obtained in S3 was filtered through a 0.22 μm filter membrane, then dialyzed through an MV: 500 dialysis membrane for 72 h, freeze-dried to form a powder, collected, and puerarin carbon dots, i.e., Pue-CDs, were obtained and stored under light-protected conditions for later use.

[0043] Detection and Analysis 1) Light conversion capability test The specific operating steps are as follows: The powdered puerarin carbon dots obtained in Example 1 were dissolved in ultrapure water to prepare a Pue-CDs aqueous solution with a concentration of 150 μg / mL. The Pue-CDs aqueous solution was then irradiated under natural light and under light with a wavelength of 360 nm. The results are as follows: Figure 1 As shown.

[0044] from Figure 1 Comparative observation shows that the Pue-CDs aqueous solution irradiated under normal light conditions is yellow (left figure), while the Pue-CDs aqueous solution irradiated under light with a wavelength of 360nm exhibits blue fluorescence, thus proving that Pue-CDs has good light conversion ability.

[0045] 2) Fluorescence intensity test The specific operating steps are as follows: The powdered puerarin carbon dots prepared in Example 1 were dissolved in ultrapure water to prepare a 150 μg / mL Pue-CDs aqueous solution. Then, 3 mL of the Pue-CDs aqueous solution was added to a fluorescence test cuvette. The fluorescence properties of the puerarin carbon dots were tested using a fluorescence spectrometer (HORIBA Corporation, Japan) at different emission wavelengths (310 nm, 330 nm, 350 nm, 370 nm, 390 nm, 410 nm, 430 nm, and 450 nm). The results are as follows: Figure 2 As shown.

[0046] from Figure 2Analysis shows that the emission spectrum of puerarin carbon dots redshifts with increasing excitation wavelength, and the emission fluorescence intensity of puerarin carbon dots is strongest after irradiation with light at a wavelength of 350 nm, thus proving that the photoconversion performance of puerarin carbon dots is best at a wavelength of 350 nm.

[0047] 3) Infrared spectroscopy test Fourier transform infrared spectroscopy was used to analyze the infrared spectra of puerarin used in S1 of Example 1 and the puerarin carbon dots (Pue-CDs) prepared in S4. The results are as follows: Figure 3 As shown.

[0048] from Figure 3 Comparative analysis shows that puerarin carbon dots (Pue-CDs) retain the functional groups of puerarin, such as phenolic hydroxyl groups, benzene rings, and ether bonds.

[0049] 4) Fluorescence test The specific operating steps are as follows: The powdered puerarin carbon dots obtained in Example 1 were dissolved in ultrapure water to prepare a 150 μg / mL Pue-CDs aqueous solution. Then, 3 mL of the Pue-CDs aqueous solution was added to a fluorescence testing cuvette. The fluorescence properties of the polyphenol carbon dots were tested using a fluorescence spectrometer (HORIBA Corporation, Japan) at different emission wavelengths (310 nm, 330 nm, 350 nm, 370 nm, 390 nm, 410 nm, 430 nm, and 450 nm). The results are as follows: Figure 4 As shown.

[0050] from Figure 4 Analysis shows that when Pue-CDs aqueous solution is irradiated with excitation light of wavelength 350nm, it emits blue light of 460nm.

[0051] 5) Particle size test The particle size of the puerarin carbon dots (Pue-CDs) prepared in Example 1 was analyzed using DLS (Dynamic Light Scattering), and the results are as follows: Figure 5 As shown.

[0052] from Figure 5 Analysis shows that the particle size of puerarin carbon dots (Pue-CDs) is uniformly distributed, with an average particle size of about 2 nm.

[0053] 6) Scanning electron microscopy test The morphology of the puerarin carbon dots (Pue-CDs) prepared in Example 1 was analyzed using scanning electron microscopy (SEM), and the results are as follows. Figure 6 As shown.

[0054] from Figure 6 Analysis showed that the particle size was uniform and evenly distributed, consistent with the results of DLS.

[0055] 7) Free radical scavenging ability test OH scavenging ability test The ability of puerarin carbon dots (Pue-CDs) to scavenge hydroxyl radicals was tested using the Fenton reaction.

[0056] The specific operating steps are as follows: Puerarin used in S1 of Example 1 and puerarin carbon dots (Pue-CDs) prepared in S4 were dissolved in PBS buffer to prepare Puerarin PBS buffer solutions and Pue-CDs PBS buffer solutions with concentrations of 10 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL, and 125 μg / mL, respectively. Then, ethanol, salicylic acid, and FeSO4 were added sequentially to wells of a plate, followed by different concentrations of Puerarin aqueous solution or Pue-CDs PBS buffer solution, and then H2O2 was added to obtain multiple test samples. The concentrations of FeSO4, salicylic acid, and H2O2 in the test samples were 9 mM and 9 mM, respectively. A blank control sample was also prepared, which, in addition to the Pue-CDs... Except for replacing the PBS buffer solution with the same volume of PBS buffer, all other conditions remained the same. Then, multiple test samples and blank control samples were placed together in a water bath and heated at 37 °C for 15 min. Subsequently, the absorbance values ​​of multiple samples at 510 nm were measured using a microplate reader. The •OH scavenging rate was calculated using equation (I). • OH scavenging rate (%) = [(H0 – H1) / H0] × 100% (Ⅰ) In formula (Ⅰ), H0 represents the absorbance value of the blank control sample, and H1 represents the absorbance value of the sample to be tested.

[0057] • The results of OH scavenging rate are as follows Figure 7 As shown.

[0058] Figure 7 In the graph, the horizontal axis represents the concentration of Puerarin in the Puerarin PBS buffer solution or the concentration of Pue-CDs in the Pue-CDs PBS buffer solution, and the vertical axis represents the •OH scavenging rate. Figure 7Comparative analysis shows that, at the same concentration, the ·OH scavenging ability of the puerarin carbon dots (Pue-CDs) prepared in Example 1 is significantly better than that of puerarin, thus proving that the ·OH scavenging ability is greatly improved by modifying puerarin.

[0059] ·O2 - Clearance capability test Nitroblue tetrazolium chloride (NBT) was used as a probe to determine its ability to scavenge superoxide radicals by removing polyphenol carbon dots.

[0060] The specific operating steps are as follows: Puerarin used in S1 of Example 1 and puerarin carbon dots (Pue-CDs) prepared in S4 were dissolved in PBS buffer to prepare aqueous solutions of Puerarin and Pue-CDs with concentrations of 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 75 μg / mL, respectively. Then, different concentrations of Puerarin PBS buffer solution or Pue-CDs were added... Riboflavin, methionine, NBT (nitrotetrazolium chloride), and PBS buffer (pH 7.35) were added to PBS buffer solution to obtain multiple test samples. The concentrations of riboflavin, methionine, NBT, and PBS buffer in these samples were 6.67 μM, 4.33 mM, 25 μM, and 0.01 M, respectively. An NBT volume equal to that of a single test sample was used as a blank control, and a PBS buffer volume equal to that of a single test sample was used as a negative control. The test samples, blank control, and negative control were then irradiated with a 30 W UV lamp for 10 min. The absorbance at 560 nm was then measured using a microplate reader. O2 was calculated using equation (II). - Clearance rate; ·O2 - Sweep rate = [(A1 – A2) / (A1 – A0)] × 100% (II) In formula (II), A0 represents the absorbance of the blank control sample, A1 represents the absorbance of the negative control sample, and A2 represents the absorbance of the test sample.

[0061] ·O2 - The clearance rate results are as follows Figure 8 As shown.

[0062] Figure 8 In the diagram, the horizontal axis represents the concentration of Puerarin in the Puerarin PBS buffer solution or the concentration of Pue-CDs in the Pue-CDs PBS buffer solution, and the vertical axis represents the concentration of O2.- Clearance rate. From Figure 8 Comparative analysis shows that, at the same concentration, the ·O2 of the puerarin carbon dots (Pue-CDs) prepared in Example 1 is higher. - The scavenging ability was significantly better than that of puerarin, thus proving that modification of puerarin greatly enhances the scavenging capacity of ·O2. - Clearance capability.

[0063] DPPH Scavenging Capacity Test The specific operating steps are as follows: Puerarin used in S1 of Example 1 and puerarin carbon dots (Pue-CDs) prepared in S4 were dissolved in ethanol to prepare ethanol solutions with concentrations of 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 75 μg / mL, respectively. These solutions were then incubated at 37 °C in the dark for 15 min to obtain multiple test samples. An equal volume of 2,2-diphenyl-1-picrylhydrazine (DPPH) was used as a blank control. The absorbance of the test samples and the blank control samples at 517 nm was then measured using a microplate reader. The DPPH scavenging rate was calculated using formula (III). DPPH clearance rate = [(D0 – D1) / D0] × 100% (III) In formula (Ⅲ), D0 represents the absorbance of the sample to be tested, and D1 represents the absorbance of the blank control sample DPPH.

[0064] The results of DPPH clearance rate are as follows Figure 9 As shown.

[0065] Figure 9 In the graph, the horizontal axis represents the concentration of Puerarin in the ethanol solution or the concentration of Pue-CDs in the ethanol solution, and the vertical axis represents the DPPH scavenging rate. Figure 9 Comparative analysis shows that, at the same concentration, the DPPH scavenging ability of the puerarin carbon dots (Pue-CDs) prepared in Example 1 is significantly better than that of puerarin, thus proving that the DPPH scavenging ability is greatly improved by modifying puerarin.

[0066] 8) Cell live / dead staining experiment The specific operating steps are as follows: (1) The puerarin carbon dots (Pue-CDs) prepared in Example 1 were dissolved in the culture medium (high glucose DMEM / F12 medium + 1% penicillin antibiotics + 10% fetal bovine serum) to prepare a culture medium containing Pue-CDs; then ATDC5 cells were seeded into 24-well plates at a rate of 1×10^5 cells / mL, and culture medium (high glucose DMEM / F12 medium + 1% penicillin antibiotics + 10% fetal bovine serum, 1 mL per well) was added, and then culture medium containing Pue-CDs was added to each well, so that the final concentration of Pue-CDs in the cell culture medium was 0 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 250 μg / mL, respectively. Then the cells were incubated in a cell culture incubator at 37°C with 5% CO2 air for 24 hours.

[0067] (2) Discard the culture medium and wash the cells three times with 1×PBS.

[0068] (3) Add 1 ml of PBS and 2 uL of Calcein-AM (Solepro) to each well and incubate at 37°C in the dark for 20 min.

[0069] (4) Add 1 μL of the PI stock solution provided in the kit to the cells to be stained, and stain at room temperature in the dark for 5 min.

[0070] (5) Discard the staining solution after incubating the cells with fluorescence.

[0071] (6) Wash the cells three times with 1x PBS.

[0072] (7) Live cells were detected using a 490nm excitation filter (green fluorescence) and dead cells were detected using a 560nm emission filter under a confocal microscope (red fluorescence). Results are as follows: Figure 10 As shown.

[0073] Figure 10 In the graph, the horizontal axis represents the concentrations of Pue-CDs in the cell culture medium, with 0 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 250 μg / mL representing the concentrations of Pue-CDs in the cell culture medium, respectively. The vertical axis represents the images of green live cells viewed through the FITC channel, the images of red dead cells viewed through the RFP channel, and the images of green live cells viewed through the FITC channel and red dead cells viewed through the RFP channel merged.

[0074] from Figure 10The AMPI results showed that within a certain concentration range (50~250 μg / mL), Pue-CDs maintained green fluorescence and did not increase red fluorescence, indicating that puerarin carbon dots have no obvious toxicity and that Pue-CDs did not increase cell death when stimulating cells at gradient concentrations.

[0075] 9) CCK-8 cell viability / toxicity colorimetric assay The specific operating steps are as follows: S1. Add 100 μL (5000-10000 cells) of ATDC-5 (mouse chondrocyte) cells and culture medium (high glucose DMEM / F12 medium + 1% penicillin antibody + 10% fetal bovine serum, 200 μL per well) to each well of a 96-well plate as the test sample. Leave two wells without cells and add the same volume of culture medium (high glucose DMEM / F12 medium + 1% penicillin antibody + 10% fetal bovine serum, 200 μL per well) as blank control samples. Then, incubate the plates in a cell culture incubator at 37°C with 5% CO2 air for 24 h. S2. The puerarin carbon dots (Pue-CDs) prepared in Example 1 were dissolved in culture medium (high glucose DMEM / F12 medium + 1% penicillin antibiotics + 10% fetal bovine serum) to prepare a culture medium containing Pue-CDs. Then, culture medium containing Pue-CDs was added to each well in sequence, so that the final concentrations of Pue-CDs in the cell culture medium were 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 250 μg / mL of Pue-CDs aqueous solution, respectively, for stimulation. Two control groups were not given Pue-CDs, but were given the same volume of culture medium (high glucose DMEM / F12 medium + 1% penicillin antibiotics + 10% fetal bovine serum, 200 μL per well). S3. Place the 96-well plate in a cell culture incubator at 37°C with 5% CO2 air and 100% humidity for 24 hours. S4. Add 10 μL of CCK-8 solution to each well, and then incubate in an incubator at 37°C with 5% CO2 air for 24 h or 48 h to obtain the incubated test sample and the incubated blank control sample. S5 and a fluorescence microplate reader were used to measure the absorbance of the test sample and the blank control sample at 450 nm after incubation.

[0076] Cell viability: Subtract the background OD value (OD value of the blank control sample) from the OD value of each test sample. The OD value of each replicate well is taken as the average ± SD. Cell viability is calculated using Equation (Ⅳ). Cell viability % = (OD value of the sample to be tested after incubation / OD value of the blank control sample after incubation) × 100%.

[0077] Cell survival results as follows Figure 11 As shown.

[0078] Figure 11 The left image shows the CCK-8 incubation results after 24 hours, and the right image shows the results after 48 hours. The horizontal axis represents the final concentration of Pue-CDs in the cell culture medium, and the vertical axis represents the cell viability. Figure 11 Comparative analysis showed that Pue-CDs were not toxic to cells at different concentrations.

[0079] 10) Fluorescence intensity emitted by Pue-CDs solutions after irradiation with 360nm light in H2O2 solutions of different concentrations. The specific operating steps are as follows: S1. Add 100 μL of H2O2 aqueous solution with concentrations of 0%, 5%, 10%, 15%, 20%, 25%, and 30% to a 96-well plate in sequence, with 5 groups for each concentration; S2. Add 50 μL of an aqueous solution of puerarin carbon dots (Pue-CDs) with a concentration of 100 μg / mL, prepared from the puerarin carbon dots (Pue-CDs) obtained in Example 1, to each group of solutions. S3. Measure the fluorescence intensity of each well using a fluorescence microplate reader and plot the correlation curve using GraphPad Prism.

[0080] The results are as follows Figure 12 As shown.

[0081] Figure 12 In the diagram, the horizontal axis represents the concentration of H2O2 in the H2O2 aqueous solution, and the vertical axis represents the fluorescence intensity. Figure 12 Analysis revealed that the intensity of the 460nm emission light generated by Pue-CDs under 360nm excitation light irradiation was linearly related to the fluorescence intensity of ROS. As the H2O2 concentration increased, Pue-CDs were consumed, and the upconversion fluorescence weakened. This demonstrates that the puerarin carbon dots of this invention possess signal output capability and can simultaneously monitor and provide feedback on ROS concentration changes and therapeutic efficacy during treatment, achieving effective integration of "treatment-monitoring."

[0082] 11) ROS fluorescence intensity of ATDC-5 after intervention with different Pue-CDs concentrations and IL-1β stimulation The specific operating steps are as follows: Cell culture: ATDC-5 cells and culture medium (high glucose DMEM / F12 medium + 1% penicillin antibiotics + 10% fetal bovine serum) were added to 48-well plates. Then, IL-1β was added to each well so that the concentration of IL-1β in the cell culture medium was 10 ng / mL. Subsequently, culture medium containing puerarin carbon dots (Pue-CDs) prepared in Example 1 (high glucose DMEM / F12 medium + 1% penicillin antibiotics + 10% fetal bovine serum) was added to each well sequentially so that the final concentrations of Pue-CDs in the culture medium were 0 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, and 250 μg / mL, respectively, to stimulate the cells. The cells were then cultured in a 5% CO2 cell culture incubator at 37°C for 24 h and the cells were collected.

[0083] After cell collection, the probe (Beyotime Reactive Oxygen Spectrometry Kit) was loaded: DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) was diluted with PBS buffer at a volume ratio of 1:1000 to achieve a final concentration of 10 μmol / L. Cells were collected and suspended in the diluted DCFH-DA solution, and incubated at 37ºC for 20 minutes. The cells were inverted and mixed every 3-5 minutes to ensure thorough contact between the probe and cells. Cells were washed three times with PBS to remove any uninfiltrated DCFH-DA. Detection: The ROS fluorescence intensity generated by ATDC-5 after stimulation (488nm excitation wavelength, 525nm emission wavelength) was measured using a fluorescence spectrophotometer. The results are as follows: Figure 13 As shown.

[0084] The results are as follows Figure 13 As shown.

[0085] Figure 13 In the graph, the horizontal axis represents the concentration of Pue-CDs in the cell culture medium, and the vertical axis represents the ROS fluorescence intensity. From... Figure 13 Analysis showed that as the concentration of Pue-CDs increased, the intensity of ROS fluorescence generated by ATDC-5 after IL-1β stimulation decreased, thus proving that Pue-CDs can play a role in scavenging reactive oxygen species and anti-inflammation in cells.

[0086] 12) NF-κb immunofluorescence staining test The specific operating steps are as follows: S1. Cell Culture: ATDC-5 cells were added to confocal dishes as a control, an IL-1β stimulation group (IL-1β), and an IL-1β stimulation + Pue-CDs intervention group (Pue-CDs) (IL-1β 10 ng / ml, Pue-CDs 50 ug / ml). The control group received no drugs. The IL-1β stimulation group received 10 ng / mL of IL-1β (interleukin-1β) to achieve an IL-1β concentration of 10 ng / ml in the cell culture medium. The IL-1β stimulation + Pue-CDs intervention group received both IL-1β and Pue-CDs prepared in Example 1 to achieve an IL-1β concentration of 10 ng / ml and a Pue-CDs concentration of 50 ug / mL in the cell culture medium. All groups were then incubated at 37°C in a cell culture incubator containing 5% CO2 air for 24 h. S2. Sample processing: After washing with PBS 3 times, fix with 4% paraformaldehyde for 20 minutes. After fixation, wash with PBS 3 times.

[0087] S3, Permeability: Permeate with 0.1% Triton X-100 PBS for 30 minutes, then wash with PBS.

[0088] S4. Blocking: Block with 2% BSA at room temperature for 30 minutes.

[0089] S5. Dilute the NF-κb primary antibody with 2% BSA blocking buffer and incubate overnight at 4°C. The next day, wash three times with PBST and incubate with the secondary antibody in the dark. Finally, stain with DAPI (1ug / ml) for 15 minutes.

[0090] Wash three times with S6 and PBS, mount with anti-quenching mounting medium, and observe the image under a confocal microscope.

[0091] The results are as follows Figure 14 As shown.

[0092] Figure 14 In the graph, the vertical axis represents the control group, IL-1β represents the IL-1β stimulation group, and Pue-CDs represents the IL-1β+Pue-CDs stimulation group. The horizontal axis represents the graph of all color combinations of the control group, IL-1β stimulation group, and IL-1β+Pue-CDs stimulation group. NF-kb represents the green fluorescence of the control group, IL-1β stimulation group, and IL-1β+Pue-CDs stimulation group, and Dapi represents the blue fluorescence of the control group, IL-1β stimulation group, and IL-1β+Pue-CDs stimulation group.

[0093] from Figure 14Analysis showed that Pue-CDs intervention on ATDC-5 after IL-1β stimulation could effectively inhibit the expression and nuclear translocation of NF-κb, thus proving that Pue-CDs have a significant anti-inflammatory effect.

[0094] 13) ROS testing The specific operating steps are as follows: S1. Cell Culture: ATDC-5 cells were added to confocal dishes as control, IL-1β stimulation, Pue-CDs intervention, and IL-1β stimulation + Pue-CDs intervention (IL-1β 10 ng / ml, Pue-CDs...). The control group (control-control) received no drugs, the IL-1β stimulation group (IL-1β-control) received IL-1β (interleukin-1β) to make the concentration of IL-1β in the cell culture medium 10 ng / ml, the Pue-CDs intervention group (Pue-CDs-control) received Pue-CDs prepared in Example 1 to make the concentration of Pue-CDs in the cell culture medium 50 ug / mL, and the IL-1β stimulation + Pue-CDs intervention group (IL-1β-Pue-CDs) received both IL-1β (interleukin-1β) and Pue-CDs prepared in Example 1 to make the concentration of IL-1β in the cell culture medium 10 ng / ml and the concentration of Pue-CDs 50 ug / mL. Then, each group was placed in a cell culture incubator at 37°C with 5% CO2 air for 24 h.

[0095] S2. Loading the probe (Beyotime Reactive Oxygen Detection Kit): Dilute DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) with PBS at a ratio of 1:1000 to a final concentration of 10 μmol / L. Remove the cell culture medium and add an appropriate volume of diluted DCFH-DA. The volume added should be sufficient to completely cover the cells. Incubate at 37ºC for 20 minutes. Wash the cells three times with PBS to thoroughly remove any DCFH-DA that has not yet entered the cells.

[0096] S3. Detection: Direct observation using a laser confocal microscope (488nm excitation wavelength, 525nm emission wavelength).

[0097] The results are as follows Figure 15 As shown.

[0098] Figure 15In the diagram, the vertical axis represents the control group, the horizontal axis represents the IL-1β stimulation group, the vertical axis represents the Pue-CDs intervention group, and the vertical axis represents the IL-1β stimulation + Pue-CDs intervention group.

[0099] from Figure 15 Analysis showed that adding Pue-CDs did not increase intracellular ROS, and intervention with Pue-CDs on IL-1β-stimulated ATDC-5 could effectively scavenge intracellular reactive oxygen species.

[0100] In summary, the puerarin carbon dots of this invention, by using puerarin as a carbon source, enable the phenolic hydroxyl groups, carboxyl groups, and conjugated π domains on the surface of the polyphenol carbon dots to form an integrated "shell-drug" complex with the isoflavone skeleton of puerarin through the synergistic effect of hydrogen bonding-π stacking. This allows the carbon dots to systematically solve the bottlenecks of poor water solubility, easy degradation, and low bioavailability of puerarin through the five-in-one principle of "solubilization-stabilization-long-cycle-tracing-metabolic inhibition". It also enables the carbon dots to not only emit light but also possess the therapeutic function of puerarin, laying a platform for its nano-diagnostic and therapeutic applications.

[0101] The method for synthesizing puerarin carbon dots of the present invention utilizes the temperature difference-driven self-assembly of the "m-phenylenediamine-puerarin" binary system in an inorganic solvent: during the heating stage, m-phenylenediamine pre-organizes with the puerarin isoflavone skeleton through π-π stacking to form a homogeneous molecular complex; during the cooling stage, m-phenylenediamine undergoes mild oxidation-polymerization in a weakly acidic medium to generate a polyphenolic carbon dot shell rich in phenolic hydroxyl groups in situ, while the hydrogen bond network tightly embeds puerarin in a <5 nm core-shell structure. The entire process requires no strong alkali, metal catalysts, or high-energy ultrasound, avoiding the oxidative breakage of the C-4′ hydroxyl group and C-ring double bond of puerarin, thus preserving its antioxidant and NF-κB inhibitory activities. The low-temperature nucleation and slow growth mechanism results in a particle size distribution of <10 nm and a PDI <0.15, allowing direct glomerular filtration and achieving "zero residue" renal clearance. The m-phenylenediamine polymer shell possesses a natural phenolic network, endowing the nanomedicine with excellent water solubility and ROS scavenging ability, synergistically reducing the toxicity of the carrier itself. This provides a green, scalable, and universal platform for the high-activity, low-toxicity nanomedicine applications, and has significant potential for widespread application in the field of pharmaceutical technology.

[0102] The above embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

Claims

1. A puerarin carbon dot, characterized in that, The puerarin carbon dots were synthesized using puerarin as the carbon source and m-phenylenediamine as the nitrogen source.

2. The puerarin carbon dots according to claim 1, characterized in that, The particle size of the carbon dots in the puerarin is 2-6 nm.

3. A method for synthesizing puerarin carbon dots as described in any one of claims 1 to 3, characterized in that, Includes the following steps: Puerarin was dispersed in an inorganic solvent to obtain solution A; Dissolve m-phenylenediamine in an inorganic solvent to obtain solution B; Solution B is added to solution A, the mixture is heated to react, cooled, and filtered to obtain the filtrate. The filtrate was filtered using a filter membrane, then dialyzed, and freeze-dried to obtain puerarin carbon dots.

4. The method for synthesizing puerarin carbon dots according to claim 3, characterized in that, The heating reaction is carried out at a temperature of 170~200℃ for 6~12 hours.

5. The method for synthesizing puerarin carbon dots according to claim 3, characterized in that, The ratio of puerarin to m-phenylenediamine is 2.5 mmol: 1.2 mmol; And / or, the inorganic solvent is selected from water.

6. The method for synthesizing puerarin carbon dots according to claim 3, characterized in that, The filtrate was filtered using a 0.22 μm filter membrane; And / or, dialyze the filtrate using an MV: 500 dialysis membrane for 70-74 hours.

7. The method for synthesizing puerarin carbon dots according to claim 3, characterized in that, The prepared puerarin carbon dots emit fluorescence at a wavelength of 460 nm when irradiated with light at a wavelength of 350 nm. And / or, the prepared puerarin carbon dots emit fluorescence at a wavelength of 460 nm when irradiated with light at a wavelength of 645 nm. And / or, the prepared puerarin carbon dots have ROS scavenging and fluorescence feedback functions.

8. The application of puerarin carbon dots prepared by the synthesis method according to any one of claims 3 to 7 in the preparation of integrated diagnostic and therapeutic reagents for diseases.

9. The application according to claim 8, characterized in that, The diseases mentioned include at least one of osteoarthritis, acute lung injury, atopic dermatitis, allergic dermatitis, acne, and urticaria.

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