Glycyrrhiza source fluorescent carbon quantum dots and preparation method and application thereof

Abstract

The application discloses licorice source fluorescent carbon quantum dots and a preparation method and application thereof, and the preparation method comprises the following steps: crushing dried licorice root stems, adding water in a proportion of 100 mg to (10-20) mL, carrying out hydrothermal reaction at 140-180 DEG C for 5-6 h, filtering through a 0.22 mu m microporous filter after centrifugation, and obtaining a brown-yellow carbon quantum dot aqueous solution with special fragrance and fluorescent characteristics, which is the licorice source fluorescent carbon quantum dots. Compared with a traditional method of extracting licorice extraction liquid, the licorice source fluorescent carbon quantum dots can not only achieve significant inhibition of growth of Porphyromonas gingivalis and Staphylococcus aureus at a low concentration, but also have significant fluorescent characteristics, can realize real-time visualization of a treatment area visible to the naked eye under excitation of common handheld ultraviolet light or blue light sources, thereby improving administration accuracy, and significantly reducing plaque aggregation on a tooth surface and inhibiting gingival inflammation.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biomedical technology and nanomaterials technology, and in particular relates to a licorice-derived fluorescent carbon quantum dot, its preparation method and application. Background Technology

[0002] Porphyromonas gingivalis and Staphylococcus aureus are key pathogens in the occurrence and development of periodontitis and periodontal purulent infections. They not only directly lead to periodontal tissue destruction and tooth loss, but also are closely related to the occurrence and progression of various systemic diseases through the blood, swallowing, and immune system. Periodontitis can exacerbate the difficulty of blood sugar control in diabetes, promote the pathological development of Alzheimer's disease, and trigger or aggravate systemic diseases such as enteritis, seriously affecting the overall health level of the public and causing a heavy medical burden.

[0003] Currently, clinical treatment for periodontitis and purulent periodontal infections primarily relies on chemically synthesized drugs and antibiotics, such as chlorhexidine and metronidazole. However, long-term use can easily lead to the emergence of drug-resistant strains, oral microecological imbalance, and even cause changes in taste, tooth discoloration, and potential systemic side effects. In contrast, plant-derived natural extracts have significant advantages such as wide availability, good biocompatibility, low likelihood of inducing drug resistance, and fewer side effects, making them an important direction for the research and development of novel oral antibacterial agents.

[0004] In recent years, the rapid development of nanotechnology has provided new strategies for enhancing the bioactivity and therapeutic performance of natural products. In particular, plant-derived carbon quantum dots obtained through green synthesis methods, through the nanocarrier effect and surface functionalization modification, efficiently concentrate the active ingredients of plants. They not only possess good water dispersibility, low toxicity, and excellent biocompatibility, but also exhibit tunable fluorescence emission characteristics, high specific surface area, and ease of surface functionalization, showing great potential in imaging tracking and drug delivery. Applying them to periodontal local treatment can utilize their fluorescence properties to achieve real-time visualization of the drug delivery site and precise control of the treatment area, thereby improving the accuracy and efficacy of treatment and reducing potential impacts on surrounding healthy tissues, demonstrating promising translational prospects.

[0005] Therefore, developing a plant-based drug with fluorescent tracing capabilities that can effectively inhibit periodontal pathogens not only promises to provide a safe, precise, and effective new treatment strategy for periodontitis and related oral diseases, but also aligns with the current development trend of green bionanomedicine and precision medicine. Summary of the Invention

[0006] The purpose of this invention is to provide a licorice-derived fluorescent carbon quantum dot, its preparation method, and its application. Compared with licorice extract obtained by traditional methods, the licorice-derived fluorescent carbon quantum dots of this invention can not only significantly inhibit the growth of Porphyromonas gingivalis and Staphylococcus aureus at low concentrations, but also have significant fluorescence properties. They can be visualized in real time under common handheld ultraviolet or blue light sources, thereby improving the accuracy of drug delivery, significantly reducing plaque accumulation on the tooth surface, and inhibiting gingival inflammation.

[0007] To achieve the above objectives, this application adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing licorice-derived fluorescent carbon quantum dots, comprising the following steps: pulverizing dried licorice rhizomes, adding water at a ratio of 100 mg: (10-20) mL, carrying out a hydrothermal reaction at 140-180℃ for 5-6 h, centrifuging, and filtering through a 0.22 μm microporous membrane to obtain a brownish-yellow aqueous solution of carbon quantum dots with a special aroma and fluorescent properties, which is the licorice-derived fluorescent carbon quantum dots.

[0009] Secondly, the present invention provides licorice-derived fluorescent carbon quantum dots prepared by the above-described preparation method.

[0010] Thirdly, the present invention provides a medicament for the prevention or treatment of periodontal pathogenic bacterial infections, which is prepared from the above-mentioned licorice-derived fluorescent carbon quantum dots.

[0011] In the above technical solution, the licorice-derived fluorescent carbon quantum dots are diluted with physiological saline to prepare the drug.

[0012] In the above technical solutions, the concentration of the licorice-derived fluorescent carbon quantum dots in the drug is 25-500 μg / mL.

[0013] In the above technical solutions, the drug is locally injected or applied to the infected area.

[0014] Fourthly, the present invention provides the application of the above-mentioned licorice-derived fluorescent carbon quantum dots in the preparation of medicaments for the prevention or treatment of diseases caused by periodontal pathogenic bacteria infection.

[0015] In the above technical solutions, the disease is periodontitis or periodontal purulent infection.

[0016] In the above technical solutions, the pathogenic bacteria include Porphyromonas gingivalis and Staphylococcus aureus.

[0017] In the above technical solutions, the licorice-derived fluorescent carbon quantum dots inhibit the growth of periodontal pathogens, destroy the morphological structure of bacteria, inhibit biofilm formation, or directly kill bacteria at least one of the following:

[0018] Compared with the prior art, the outstanding advantages of the present invention are reflected in the following aspects:

[0019] This invention uses natural licorice as raw material to synthesize carbon quantum dots with strong fluorescence emission characteristics via a hydrothermal method. Compared with licorice extract prepared by traditional methods, this preparation process is simple, low-cost, and environmentally friendly, significantly improving the bioavailability of licorice's active ingredients, and also possesses fluorescence tracer function.

[0020] Experimental results show that the licorice-derived fluorescent carbon quantum dots of this invention can emit strong fluorescence visible to the naked eye under 440 nm excitation, which can be used for real-time, in-situ tracking of drug distribution in the periodontal region. Figure 2 , Figure 3 This significantly improves the precision of treatment. Colony plate experiments confirmed that the licorice-derived fluorescent carbon quantum dots of this invention, compared to traditional licorice extract, are more effective at inhibiting the growth of *Porphyromonas gingivalis* and *Staphylococcus aureus*. Figure 4 , Figure 5 Animal experiments further showed that, after treatment with the licorice-derived fluorescent carbon quantum dots of this invention, plaque accumulation on the tooth surface of periodontitis mice was significantly inhibited. Figure 6 This indicates that it has a good antimicrobial plaque formation effect. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0022] Figure 1 This is a diagram showing the strong fluorescence effect of the licorice-derived fluorescent carbon quantum dots of this invention under 440nm excitation light;

[0023] Figure 2 This is the fluorescence emission spectrum of the licorice-derived fluorescent carbon quantum dots of the present invention;

[0024] Figure 3 This is an experimental diagram showing the antibacterial effect of the licorice-derived fluorescent carbon quantum dots of this invention against Porphyromonas gingivalis and Staphylococcus aureus.

[0025] Figure 4 This is a visualization of the effect of the licorice-derived fluorescent carbon quantum dots of the present invention on periodontitis in mice;

[0026] Figure 5 This is an image showing the effect of licorice-derived fluorescent carbon quantum dots in inhibiting plaque on the surface of mouse teeth according to the present invention;

[0027] Figure 6 This is an experimental diagram showing the bactericidal effect of the licorice-derived fluorescent carbon quantum dots of this invention compared to traditional methods. Detailed Implementation

[0028] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0029] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0032] This invention provides a method for preparing licorice-derived fluorescent carbon quantum dots, comprising the following steps: pulverizing dried licorice rhizomes, adding water at a ratio of 100 mg: (10-20) mL, carrying out a hydrothermal reaction at 140-180℃ for 5-6 h, centrifuging, and filtering through a 0.22 μm microporous membrane to obtain a brownish-yellow aqueous solution of carbon quantum dots with a special aroma and fluorescent properties, which is the licorice-derived fluorescent carbon quantum dots of this invention.

[0033] The preferred ratio of pulverized licorice root to water is 100 mg: 20 mL. The preferred temperature for the hydrothermal reaction is 160 °C, and the preferred reaction time is 5 h. The preferred centrifugation speed is 10,000 rpm, and the preferred centrifugation time is 1 h.

[0034] The licorice-derived fluorescent carbon quantum dots of this invention can emit green fluorescence with the strongest peak at about 542 nm under 440 nm excitation light, exhibiting excellent visible fluorescence properties and suitable for in vivo and in vitro fluorescence tracing.

[0035] The present invention also provides a drug for the prevention or treatment of periodontal pathogenic bacterial infection, which is prepared from licorice-derived fluorescent carbon quantum dots.

[0036] The preparation method of the drug is as follows: dilute the licorice-derived fluorescent carbon quantum dots with physiological saline to prepare the drug of the present invention.

[0037] Preferably, the concentration of licorice-derived fluorescent carbon quantum dots in the drug is 25-500 μg / mL, and more preferably 250 μg / mL.

[0038] Preferably, the drug is injected locally or applied topically to the infected area.

[0039] This invention also provides the application of licorice-derived fluorescent carbon quantum dots in the preparation of medicaments for the prevention or treatment of diseases caused by periodontal pathogenic bacteria.

[0040] Preferably, the disease is periodontitis or purulent periodontal infection.

[0041] Preferably, the pathogenic bacteria include Porphyromonas gingivalis and Staphylococcus aureus.

[0042] The present invention utilizes licorice-derived fluorescent carbon quantum dots to inhibit the growth of periodontal pathogens, disrupt bacterial morphology and structure, inhibit biofilm formation, or directly kill at least one of the following:

[0043] This invention utilizes licorice-derived fluorescent carbon quantum dots to inhibit the growth of periodontal pathogens, specifically manifested as a significant decrease in colony count on agar plates (see [link]). Figure 4 , Figure 5 ).

[0044] This invention relates to licorice-derived fluorescent carbon quantum dots for local injection or application in the treatment of periodontitis; during application, fluorescence can be excited in real time using ultraviolet or blue light sources, allowing for precise monitoring of drug distribution and avoiding impact on surrounding healthy tissues (see [link]). Figure 2 , Figure 3 ).

[0045] Example: Preparation of fluorescent antibacterial agent (CDs-1-9) under preferred process parameters

[0046] The dried licorice root and stem were pulverized, and 100 mg was weighed out and added to ultrapure water. The mixture was then transferred to a 50 mL high-pressure reactor lined with polytetrafluoroethylene (PTFE). The reactor was placed in an oven and reacted at high temperature for several hours. After the reactor cooled naturally to room temperature, the reaction solution was removed and filtered using a 0.22 μm microporous membrane. The resulting brownish-yellow licorice-derived fluorescent carbon quantum dot stock solution with a distinctive aroma was stored at 4 °C for later use.

[0047] By precisely controlling the hydrothermal temperature (140-180℃), the volume of ultrapure water (10-20mL), and the reaction time (5-6h), the obtained products were sequentially labeled as CDs-1, CDs-2, CDs-3, CDs-4, CDs-5, CDs-6, CDs-7, CDs-8, and CDs-9. The specific preparation parameters are shown in Table 1.

[0048] Figure 1 The test showed strong fluorescence of CDs-1 (160℃, 20mL, 5h) under 440nm excitation light. Figure 2 This is a fitting diagram of the fluorescence emission spectrum peaks of CDs-1.

[0049] Comparative Example 1: Preparation of licorice extract under other reaction temperatures, times, and solid-liquid ratios (CDs-10-14)

[0050] As described above, dried licorice root powder was used as raw material (fixed dosage 100mg). By precisely controlling the hydrothermal temperature to be below 140℃ or above 180℃, the volume of ultrapure water to be greater than 20mL, and the reaction time to be less than 5h, the resulting products were labeled as CDs-10, CDs-11, CDs-12, CDs-13, and CDs-14, respectively. The remaining steps were the same as before.

[0051] Comparative Example 2: Preparation of Licorice Extract by Traditional Method

[0052] A licorice extract prepared according to the traditional method listed in the Chinese Pharmacopoeia was used as a positive control. Dried licorice rhizomes were pulverized to a size of 0.45 mm, dried at 80°C, and then added to distilled water at a material-to-liquid ratio of 1:15 (g / mL). The extract was then extracted in a constant temperature water bath at 80°C for 2 hours. Finally, the extract was refrigerated at 4°C for 24 hours, centrifuged at 8000 rpm for 15 minutes, and filtered through a 0.22 μm membrane to obtain a clear and stable licorice extract.

[0053] Application Example 1: Comprehensive Evaluation of the Antibacterial Properties and Fluorescence Visualization Characteristics of Licorice Extract

[0054] To systematically evaluate the effects of the above-mentioned preparation process parameters (preparation method, reaction temperature, material-liquid ratio, and time) on the antibacterial activity and fluorescence properties of licorice extract, the minimum inhibitory concentration (MIC) and fluorescence spectroscopy characterization were performed on the samples (CDs-1 to CDs-14, and traditional licorice extract) prepared in the examples and comparative examples, in order to screen preparation schemes that have both excellent antibacterial effects and good fluorescence tracer capabilities.

[0055] Using *Porphyromonas gingivalis* ATCC 33277 and *Staphylococcus aureus* ATCC 29213 as test strains, samples were serially diluted with BHI liquid medium at concentration gradients of 1000, 500, 250, 100, 50, 25, 5, and 0.5 μg / mL. 50 μL of each dilution was then mixed with an equal volume of bacterial suspension (1 × 10⁻⁶). 5 CFU / mL was added to 96-well plates, with a blank control of culture medium and a bacterial growth control. Pg was anaerobically cultured at 37℃ for 72 h, and Sa was aerobically cultured at 37℃ for 24 h. Then, 10 μL of resveratrol was added to each well to indicate bacterial growth. The lowest concentration at which no color change was observed visually (i.e., no bacterial growth) was defined as the MIC value. Furthermore, the fluorescence characteristics of each sample under 440 nm excitation light were characterized using fluorescence spectroscopy.

[0056] The experimental results are shown in Table 1:

[0057] (1) The extract prepared by traditional methods consumes a large amount of licorice and does not have visible fluorescence.

[0058] (2) Licorice carbon quantum dots prepared at temperatures between 140°C and 180°C have good fluorescence intensity and antibacterial activity. When the temperature is raised to above 200°C or lowered to 120°C, the emission wavelength shifts to the non-visible region, and the fluorescence intensity decreases significantly, and the antibacterial activity also decreases.

[0059] (3) When the volume of ultrapure water increased from 10 mL to 30 mL, both fluorescence intensity and antibacterial activity showed a trend of first increasing and then decreasing. 100 mg: 20 mL was the optimal material-liquid ratio, under which the best balance between fluorescence intensity and antibacterial activity was achieved.

[0060] (3) The reaction may not be complete after 4 hours, and the performance may not be optimal. Extending the reaction to 5 hours will result in a complete reaction and peak performance. Extending the reaction to 6 hours will result in a slight decrease in fluorescence intensity and antibacterial activity.

[0061] Based on the antibacterial and fluorescence properties of carbon quantum dots under different preparation parameters, CDs-1 (160℃, 100mg: 20mL, 5h) was determined to be the optimal preparation scheme. The carbon quantum dots prepared under this condition not only showed the strongest inhibitory activity against periodontal pathogens (MIC=20-50μg / mL), but also had the strongest green fluorescence, which was easily observed by the naked eye under a common handheld UV lamp, providing a basis for the precise tracing of the distribution of antibacterial drugs in periodontal pockets.

[0062] Table 1. Comprehensive evaluation of the antibacterial properties and fluorescence visualization characteristics of licorice extract.

[0063]

[0064] Application Example 2: Bactericidal effect of optimized process fluorescent antibacterial agent (plate colony count)

[0065] The preferred samples CDs-1 (500, 250, 100, 50, 25 μg / mL) determined in the examples were co-cultured with Pg and Sa for 24 h. After the incubation, 100 μL of bacterial culture was taken from the co-culture system and serially diluted 10-fold, plated on BHI blood agar plates (Pg) or LB agar plates (Sa), and incubated at 37°C for 48 hours anaerobic and aerobic, respectively, to count colony-forming units (CFU). The experiment was repeated three times, and the data are expressed as mean ± standard deviation. Statistical analysis was performed using a t-test.

[0066] The results are as follows Figure 3 As shown, the number of Pg and Sa colonies in the experimental group treated with licorice carbon quantum dots was significantly lower than that in the control group (P<0.01, P<0.001). The bactericidal rate increased with increasing concentration, but there was no statistically significant difference between the 500 μg / mL and 250 μg / mL groups. Although licorice extract exhibited antibacterial activity ( Figure 6However, its effect was significantly lower than that of the preferred sample CDs-1 (250 μg / mL, P<0.05) at the same concentration, with a sterilization rate of less than 10%.

[0067] Application Example 3: Visualized Therapeutic Application of Optimized Fluorescent Antibacterial Agents in Periodontitis

[0068] Eight-week-old C57BL / 6 mice were used to establish a periodontitis model by ligating the right maxillary second molar with 5-0 silk suture. Starting on day 5 post-ligation, mice in the experimental group were injected with 20 μL of the preferred sample CDs-1 (250 μg / mL) into the gingival sulcus of the maxillary second molar under ultraviolet light irradiation, while the control group was injected with an equal volume of PBS. This was repeated once daily for 6 to 10 days. Immediately after each injection, the treated area was irradiated with a handheld fluorescent flashlight at 440 nm. A clear green fluorescent signal was observed, indicating the drug distribution area. Figure 4 ).

[0069] Application Example 4: Inhibitory effect of optimized process fluorescent antibacterial agent on plaque accumulation on mouse dental surfaces.

[0070] Dental plaque was stained using 0.5% erythrosine B (Sigma-Aldrich, USA) staining solution. After staining at 37°C in the dark for 5 minutes, excess stain was gently rinsed off with PBS. Images were acquired under a stereomicroscope, and the percentage of plaque stained area on the molar surface was calculated using ImageJ software. The experiment was repeated six times. Data are expressed as mean ± standard deviation and statistically analyzed using a t-test. Results are as follows: Figure 5 As shown, compared with the PBS control group, plaque accumulation on the tooth surface of mice treated with the preferred sample CDs-1 was significantly inhibited (P<0.01), and the plaque coverage was reduced by about 60%, indicating that it has a good anti-plaque effect.

[0071] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing licorice-derived fluorescent carbon quantum dots, characterized in that: Includes the following steps: The dried licorice root and stem are pulverized and added to water at a ratio of 100 mg: (15-20) mL. The mixture is subjected to a hydrothermal reaction at 140-180℃ for 5-6 hours. After centrifugation, the mixture is filtered through a 0.22 μm microporous membrane to obtain a brownish-yellow aqueous solution of carbon quantum dots with a special aroma and fluorescent properties, which is the licorice-derived fluorescent carbon quantum dots.

2. The licorice-derived fluorescent carbon quantum dots prepared by the method described in claim 1.

3. A drug for the prevention or treatment of periodontal pathogenic bacterial infections, characterized in that: It is prepared from the licorice-derived fluorescent carbon quantum dots of claim 2, wherein the periodontal pathogens include Porphyromonas gingivalis and Staphylococcus aureus.

4. The drug according to claim 3, characterized in that: The licorice-derived fluorescent carbon quantum dots were diluted with physiological saline to prepare the drug.

5. The drug according to claim 4, characterized in that: The concentration of the licorice-derived fluorescent carbon quantum dots in the drug is 25-500 μg / mL.

6. The drug according to claim 3, characterized in that: The drug is injected locally or applied to the infected area.

7. The use of the licorice-derived fluorescent carbon quantum dots of claim 2 in the preparation of medicaments for the prevention or treatment of diseases caused by periodontal pathogens, wherein the periodontal pathogens include Porphyromonas gingivalis and Staphylococcus aureus.

8. The application according to claim 7, characterized in that: The disease is periodontitis or purulent periodontal infection.

9. The application according to claim 7, characterized in that: The licorice-derived fluorescent carbon quantum dots inhibit the growth of periodontal pathogens, disrupt bacterial morphology and structure, inhibit biofilm formation, or directly kill bacteria, at least one of the following:

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