Chonglingdan carbon dots and preparation method and application thereof

Abstract

The application discloses a kind of smelly danchang dots and preparation method and application, belong to medical technical field.The preparation method of smelly danchang dot includes: smelly danchang fine powder is dispersed in distilled water, and is stirred pyrolysis on heating table;Pyrolysis product is diluted with distilled water, and supernatant is obtained by centrifugation, filter membrane filtration, dialysis, freeze-drying, to obtain carbon dot.The average particle size of obtained carbon dot is 4.51±1.04nm.The smelly danchang dot prepared in the application shows strong inhibitory activity to a variety of gram-positive bacteria (such as listeria, methicillin-resistant staphylococcus aureus MRSA) and fungi (such as Candida albicans), especially to clinically tricky drug-resistant strains, such as vancomycin-resistant enterococci (VRE) and MRSA effective, can be applied to the preparation of antibacterial drugs, antibacterial dressing, disinfectant or preservative, with the advantages of wide raw material source, simple preparation method, low cost, environment-friendly and the like.

Description

Technical Field

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

[0002] Traditional Chinese medicine resources are a valuable repository for discovering new bioactive molecules. (The text then mentions "Choulingdan" which is likely an unrelated product name.) Laggera pterodonta (DC.) Benth. is a traditional Chinese medicine used for clearing heat and detoxifying, widely employed in folk medicine to treat infectious diseases such as colds, sore throats, carbuncles, and boils. Modern pharmacological studies have shown that *Choulingdan* is rich in active ingredients such as flavonoids, sesquiterpenes, and phenolic acids, exhibiting good anti-inflammatory, antiviral, and antibacterial activities. However, the extracts of *Choulingdan* obtained by traditional water or alcohol extraction methods have limitations such as complex composition, low solubility of active ingredients, low bioavailability, and unclear mechanism of action, restricting its modern development and clinical application.

[0003] In recent years, the rise of nanotechnology has provided new directions for innovative research in traditional Chinese medicine. Carbon dots (CDs), as an emerging carbon-based nanomaterial, have attracted widespread attention due to their small size (typically less than 10 nm), good biocompatibility, low toxicity, high water solubility, ease of functionalization, and unique optical properties. Studies have shown that some carbon dots themselves or their complexes exhibit excellent antibacterial properties, and their antibacterial mechanisms may include physical disruption of bacterial cell membranes, induction of reactive oxygen species (ROS), and influence on bacterial metabolism. Currently, there are various carbon sources for the synthesis of carbon dots, but the research on preparing carbon dots with both the pharmacological activity of the parent drug and the unique properties of nanomaterials using a single, bioactive traditional Chinese medicine as a precursor through a green and simple method, and applying them to antibacterial drugs, is still in its early stages.

[0004] There are currently no reports related to carbon dots in the stinking pill. Summary of the Invention

[0005] To address the problems of existing technologies, this invention utilizes the traditional Chinese medicine Choulingdan as a carbon source to develop a simple, environmentally friendly, and low-cost carbon dot for Choulingdan. This carbon dot exhibits significant antibacterial activity against drug-resistant bacteria such as Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and Candida albicans, and holds promise as a nano-antibacterial agent, providing a new strategy and approach for solving the problem of bacterial resistance.

[0006] To achieve the objectives of this invention, the following technical solutions are provided:

[0007] First, this invention provides the application of carbon dots from *Clerodendrum trichotomum* in the preparation of antibacterial drugs, antibacterial dressings, disinfectants, or preservatives. The carbon dots are characterized by having an average particle size of 4.51 ± 1.04 nm, an absorption peak at 545 nm, and emitting green fluorescence at an excitation wavelength of 470 nm. The preparation method includes the following steps:

[0008] (1) Disperse the fine powder of stinking pill in distilled water and stir and pyrolyze at 350±10℃ to obtain the pyrolysis product;

[0009] (2) The pyrolysis product is diluted with distilled water, centrifuged to obtain the supernatant, filtered through a filter membrane, dialyzed, and freeze-dried to obtain the carbon point of the stinking pill.

[0010] Preferably, in step (1), the mass-to-volume ratio of the fine powder of the stinking pill to distilled water is 1:2-3, with units of g / ml; and the pyrolysis time is 6-7 min.

[0011] The so-called "stinky pill" (scientific name) :Laggera pterodonta Also known as lion grass or stinky leaves, it is a plant belonging to the genus *Chrysanthemum* in the family Asteraceae. The whole plant is used medicinally; it has a bitter and pungent taste, is cold in nature, and possesses various effects such as clearing heat and detoxifying, reducing inflammation and relieving pain, and eliminating phlegm and relieving cough.

[0012] Preferably, the particle size of the fine powder of *Choulingdan* in step (1) is 80-120 mesh.

[0013] Preferably, in step (2), the mass-to-volume ratio of the pyrolysis product to distilled water is 1:2 to 3, with units of g / ml.

[0014] Preferably, the centrifugation speed in step (2) is 10,000 to 12,000 rpm and the centrifugation time is 28 to 32 minutes.

[0015] Preferably, the dialysis time in step (2) is 40 to 50 hours; the drying is freeze drying, and the freeze drying time is 72 to 96 hours.

[0016] Preferably, the filter membrane in step (2) has a pore size of 0.22 μm, and the dialysis bag for dialysis has a molecular weight cutoff of 3500 Da.

[0017] Preferably, the carbon dots of the stinky elixir inhibit the activity of Gram-positive bacteria or fungi.

[0018] More preferably, the carbon dots of the stinky granules inhibit the activity of Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and Candida albicans.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. Green raw materials, widely available and inheriting traditional medicinal effects: This invention uses the traditional Chinese medicine Choulingdan as the sole carbon source, making the raw materials natural, green, and renewable. It realizes the nano-scale and high-value utilization of Chinese medicinal resources, representing a new path for the modernization of Chinese medicine research.

[0021] 2. The preparation method is extremely simple, rapid, and low-cost: This invention employs a one-step hydrothermal pyrolysis method, eliminating the need for any expensive chemical reagents, complex catalysts, or cumbersome synthesis steps. The entire reaction process is completed within minutes (only 6-7 minutes), with mild reaction conditions, simple operation, and extremely low energy consumption, making it highly suitable for large-scale industrial production and offering significant economic benefits.

[0022] 3. Excellent water solubility and high biocompatibility: The carbon dots of *Clerodendrum trichotomum* prepared by this method have a uniform particle size (4.51±1.04 nm), are rich in hydrophilic groups on the surface, and exhibit good dispersibility and high stability in aqueous solution. As it originates from a natural plant, it is expected to have low cytotoxicity and good biocompatibility, laying a solid foundation for its biomedical applications.

[0023] 4. Broad antibacterial spectrum, especially significant activity against drug-resistant strains: The carbon dots of the stinking pill obtained in this invention show strong inhibitory activity against a variety of Gram-positive bacteria (such as Listeria, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE)) and fungi (such as Candida albicans), especially effective against clinically challenging drug-resistant strains such as VRE and MRSA. This indicates that it has great potential to overcome resistance to traditional antibiotics and provides a nano-antibacterial agent option for solving the global problem of bacterial resistance.

[0024] 5. Broad Application Prospects: The carbon dots of this odorant can be further developed into antibacterial drugs, antibacterial dressings, disinfectants, or preservatives for the treatment of various infectious diseases caused by drug-resistant bacteria. Furthermore, its inherent fluorescent properties make it possible to construct integrated diagnostic and therapeutic platforms (such as simultaneous bacterial imaging and sterilization), offering diverse application value. Attached Figure Description

[0025] Figure 1 This is a high-magnification transmission electron microscope image of the carbon dots of *Lycoperdon perlatum* prepared in Example 1 of this invention;

[0026] Figure 2 This is a particle size distribution diagram of the carbon dots of *Choulingdan* prepared in Example 1 of the present invention;

[0027] Figure 3 The infrared (IR) spectrum of carbon dots of *Lycoperdon perlatum* prepared in Example 1 of this invention;

[0028] Figure 4The UV-vis image of the carbon dots of *Clerodendrum trichotomum* prepared in Example 1 of this invention;

[0029] Figure 5 The PL image shows the carbon dots of the *Choulingdan* prepared in Example 1 of this invention.

[0030] Figure 6 The image shows the XRD pattern of carbon dots of the *Choulingdan* prepared in Example 1 of this invention.

[0031] Figure 7 The image shows the X-ray photoelectron energy (XPS) spectrum of the carbon dots of *Lycoperdon perlatum* prepared in Example 1 of this invention. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0033] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0034] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0035] The following examples contain some of the main raw material sources:

[0036] Stinky Elixir ( Laggera pterodonta Collected in Dehong Prefecture, Yunnan Province.

[0037] Example 1: Preparation of carbon dots from *Clerodendrum trichotomum*

[0038] (1) Crush the stinking pill into fine powder (80-120 mesh) and disperse it in 2.5 times (mass-volume ratio) of distilled water, and stir and pyrolyze at 350℃ for 6 min to obtain the pyrolysis product;

[0039] (2) The pyrolysis product was diluted with 2.5 times (by mass / volume) of distilled water, mixed thoroughly, and centrifuged at 11,000 rpm for 30 minutes. The supernatant was collected and filtered through a 0.22 μm pore size filter membrane. The filtrate was placed in a dialysis bag (with a molecular weight cutoff of 3500 Da) and dialyzed in distilled water for 45 hours, with the dialysate replaced every 8 hours. After dialysis, the dialysate was freeze-dried for 82 hours to obtain the carbon dots of the stinking pill.

[0040] The carbon dots of the obtained *Clerodendrum trichotomum* were detected and analyzed using high-magnification transmission electron microscopy, Fourier transform infrared spectroscopy, ultraviolet-visible absorption spectroscopy, fluorescence spectrophotometry, X-ray diffraction, and X-ray photoelectron spectroscopy. The results are shown in [Figure number missing]. Figure 1-7 The results showed that the carbon dots of the stinking pill had a particle size of 4.51±1.04 nm, an absorption peak at 545 nm, and emitted green fluorescence when the excitation wavelength was 470 nm.

[0041] Example 2: Preparation of carbon dots in *Choulingdan*

[0042] (1) Crush the stinking pill into fine powder (80-120 mesh) and disperse it in 2 times (mass-volume ratio) of distilled water, and stir and pyrolyze at 340℃ for 7 min to obtain the pyrolysis product;

[0043] (2) The pyrolysis product was diluted with 3 times (by mass / volume) of distilled water, mixed thoroughly, and centrifuged at 10,000 rpm for 32 minutes. The supernatant was collected and filtered through a 0.22 μm pore size filter membrane. The filtrate was placed in a dialysis bag (with a molecular weight cutoff of 3500 Da) and dialyzed in distilled water for 40 hours, with the dialysate replaced every 8 hours. After dialysis, the dialysate was freeze-dried for 72 hours to obtain the carbon dots of the stinking pill.

[0044] Upon testing, the carbon dots obtained from the stinking pill were the same as those in Example 1.

[0045] Example 3: Preparation of carbon dots in *Choulingdan*

[0046] (1) Crush the stinking pill into fine powder (80-120 mesh) and disperse it in 3 times (mass-volume ratio) of distilled water, and stir and pyrolyze at 360℃ for 6 min to obtain the pyrolysis product;

[0047] (2) Dilute the pyrolysis product with 2 times (by mass / volume) of distilled water, mix thoroughly, centrifuge at 11,000 rpm for 28 minutes, collect the supernatant, filter it using a 0.22 μm pore size filter membrane, and put the filtrate into a dialysis bag (molecular weight cutoff of 3500 Da). Dialyze in distilled water for 50 hours, changing the dialysis fluid every 8 hours. After dialysis, freeze-dry the dialysis fluid for 96 hours to obtain the carbon dots of the cinnabar.

[0048] Upon testing, the carbon dots obtained from the stinking pill were the same as those in Example 1.

[0049] Comparative Example 1

[0050] The difference from Example 1 is that the pyrolysis temperature in step (1) is 250°C, while the rest is the same as in Example 1.

[0051] Comparative Example 2

[0052] The difference from Example 1 is that the pyrolysis temperature in step (1) is 300°C, while the rest is the same as in Example 1.

[0053] Comparative Example 3

[0054] The difference from Example 1 is that the pyrolysis temperature in step (1) is 400°C, while the rest is the same as in Example 1.

[0055] The yields of carbon dots of the stinking pill obtained in Examples 1-3 and Comparative Examples 1-3 are shown in Table 1.

[0056] Table 1. Yields of carbon dots prepared from *Clerodendrum trichotomum* in Examples 1-3 and Comparative Examples 1-3

[0057]

[0058] Example 4 Antibacterial performance test

[0059] The MIC (minimum inhibitory concentration) detection method was used to select S epidermidis CMCC26069 (Staphylococcus epidermidis CMCC 26069), B. cereus CMCC63303 (Bacillus cereus CMCC 63303) E. coli s 27 (Escherichia coli S27), L. monocytogenes ATCC19114 (Listeria monocytogenes ATCC 19114), MRSA USA300 (Methicillin-resistant Staphylococcus aureus USA 300), VRE ATCC51299 (Vancomycin-resistant Enterococcus ATCC51299), C albicans Common bacteria such as ATCC 14053 (Candida albicans ATCC 14053) were used as test subjects.

[0060] The minimum inhibitory concentration (MIC) was determined using the micro-broth dilution method: the concentration of the logarithmic growth phase bacterial suspension was adjusted to 0.5 × 10⁻⁶. 5 CFU / mL was used in 96-well plates for sequential two-fold dilutions of the adjusted bacterial suspension. Specifically, the carbon dots prepared in Example 1 and Comparative Examples 1-3 were diluted to four concentration gradients (0.5, 0.25, 0.125, 0.0625 mg / mL), and the 95% ethanol extract of *Clerodendrum trichotomum* was diluted to four concentration gradients (0.5, 0.25, 0.125, 0.0625 mg / mL). Each concentration was achieved by mixing bacterial suspension and liquid carbon dots in different volumes to a total volume of 100 μL. Control wells (50 μL bacterial suspension + 50 μL sterile water) were also included, and sterile water was added to the edge wells of the 96-well plate to reduce evaporation interference. The plates were incubated at 37°C for 24 h, with six replicate wells for each sample concentration to ensure experimental accuracy. OD was then measured. 600(Except for the edge wells), observe the changes in turbidity within the wells. The minimum drug concentration required to completely inhibit bacterial growth is defined as the MIC value, and the results are shown in Table 2.

[0061] Table 2. Minimum inhibitory concentration (MIC) of carbon dots in *Clerodendrum trichotomum* prepared at different temperatures.

[0062]

[0063] Note: - indicates that the MIC is significantly higher than 512 μg / mL.

[0064] The results showed that the carbon dots prepared using the method of this invention exhibited the best antibacterial activity, demonstrating strong inhibitory activity against a variety of Gram-positive bacteria (such as Listeria and methicillin-resistant Staphylococcus aureus MRSA) and fungi (such as Candida albicans), particularly effective against clinically challenging drug-resistant strains such as vancomycin-resistant Enterococcus (VRE) and MRSA. Compared with comparative examples 1-3, this indicates that the pyrolysis temperature has a significant impact on the antibacterial activity of the carbon dots.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of carbon dots from a certain type of styrax-based drug in the preparation of antibacterial drugs or antibacterial dressings, characterized in that, The carbon dots of the *Choulingdan* have an average particle size of 4.51 ± 1.04 nm, an absorption peak at 545 nm, and emit green fluorescence at an excitation wavelength of 470 nm. The preparation method includes the following steps: (1) Disperse the fine powder of stinking pill in distilled water and stir and pyrolyze at 350±10℃ for 6-7 min to obtain the pyrolysis product; (2) The pyrolysis product is diluted with distilled water, centrifuged to obtain the supernatant, filtered through a filter membrane, dialyzed, and freeze-dried to obtain the carbon dots of the stinking pill; The carbon dots of the described styraxe inhibit the activity of Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and Candida albicans.

2. The application according to claim 1, characterized in that: The mass-to-volume ratio of the fine powder of the stinking pill to distilled water in step (1) is 1:(2-3), and the unit is g / ml.

3. The application according to claim 1, characterized in that: The particle size of the fine powder of the stinky dan in step (1) is 80-120 mesh.

4. The application according to claim 1, characterized in that: In step (2), the mass-to-volume ratio of the pyrolysis product to distilled water is 1:(2-3), with units of g / ml.

5. The application according to claim 1, characterized in that: The centrifugation speed in step (2) is 10,000 to 12,000 rpm, and the centrifugation time is 28 to 32 minutes.

6. The application according to claim 1, characterized in that: The dialysis time in step (2) is 40 to 50 hours; the freeze-drying time is 72 to 96 hours.

7. The application according to claim 1, characterized in that: The filter membrane in step (2) has a pore size of 0.22 μm, and the dialysis bag for dialysis has a molecular weight cutoff of 3500 Da.

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