A Chinese herbal medicine-based carbon nanomaterial, a preparation method and application thereof

Abstract

The present application relates to a kind of Chinese herbal medicine-based carbon nanomaterial and its preparation method and application.The present application adds Chinese herbal medicine one red into reaction kettle, adds glutathione and formamide and stirs uniformly, carries out hydrothermal reaction, then after centrifugation, dialysis, concentration purification, freeze-drying, obtains carbon nanomaterial.The Chinese herbal medicine-based carbon nanomaterial obtained in the present application has excellent photocatalytic antibacterial performance, can effectively solve the problem of bacterial drug resistance, while realizing the efficient reuse of Chinese herbal medicine resources.

Description

Technical Field

[0001] This invention relates to the field of nanobiotechnology, and in particular to a traditional Chinese medicine-based carbon nanomaterial, its preparation method, and its application. Background Technology

[0002] Microbial infections are widely recognized as one of the most critical public health problems of the 21st century, posing a significant risk to human life and imposing a heavy economic burden on patients and society. Research by the Centers for Disease Control and Prevention predicts that by 2050, the number of people dying from bacterial infections will exceed the number dying from cancer. Antibiotics have long been the first choice for eliminating and combating microorganisms; however, overuse and misuse are rapidly diminishing their effectiveness, leading to antibiotic resistance in bacteria. Furthermore, excessive stimulation further accelerates the evolution of drug-resistant bacteria, ultimately resulting in superbugs. Although new antibiotics are under continuous development, their development lags far behind the rate at which bacterial resistance emerges. In addition, biofilm formation provides bacteria with additional protection, further increasing the difficulty of eliminating infections.

[0003] Bacterial antibiotic resistance mechanisms are classified into two categories: endogenous and exogenous. Endogenous resistance mechanisms mainly include: reduced antibiotic uptake (osmotic barriers, as bacterial defense structures, can block harmful external factors by regulating permeability, thereby reducing antibiotic uptake); altered antibiotic targets (bacteria change drug target sites through gene mutations or enzyme modifications, reducing the affinity between the drug and the target, leading to antibacterial drug inactivation); antibiotic inactivation (bacteria produce inactivating enzymes, hydrolases, inactivating enzymes, or modifying enzymes through plasmid, transposon, and chromosomal gene expression, destroying the chemical structure of antibacterial drugs and rendering them inactive); and active antibiotic expulsion (bacterial active efflux systems (efflux pumps) consist of outer membrane pore proteins, inner membrane transport carriers, and connective proteins; as a class of transport proteins that can pump harmful substances out of the bacterial body, their high expression can mediate resistance and function as a bacterial detoxification pump). Exogenous resistance mechanisms are mainly related to biofilm formation. The high development costs and short effective action periods of novel antibiotics cause their development to lag behind the evolutionary rate of drug-resistant bacteria, failing to fundamentally solve the problem of bacterial resistance. Therefore, developing novel antibacterial strategies to replace traditional antibiotics for the treatment of bacterial infections is of great practical significance. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a traditional Chinese medicine-based carbon nanomaterial, its preparation method, and its application.

[0005] This invention provides a method for preparing traditional Chinese medicine-based carbon nanomaterials, comprising the following preparation steps:

[0006] S1: Add glutathione and small red dye to the reaction vessel, then add formamide, stir evenly and then react to obtain the reaction product; S2: After cooling the reaction product obtained in S1 to room temperature, first centrifuge to remove the precipitate, then dialyze the supernatant and collect the liquid in the dialysis bag. S3: The liquid in the dialysis bag is repeatedly frozen and thawed and filtered to achieve concentration and purification. Finally, it is freeze-dried to obtain traditional Chinese medicine-based carbon nanomaterials.

[0007] Furthermore, the reaction is carried out at a temperature of 140-160 °C for a time of 4-8 h.

[0008] Furthermore, the dialysis duration is 5-7 days.

[0009] Furthermore, the centrifugation speed is 5000-10000 r / min, the time is 10-20 min, and the filtration method is to use a filter membrane for filtration.

[0010] The present invention also provides a traditional Chinese medicine-based carbon nanomaterial, which is prepared according to the method described above.

[0011] Furthermore, the nanomaterials are smaller than 10 nm in size and have a large specific surface area, allowing them to adsorb onto the surface of bacteria, affecting their exchange of substances and information with the outside world and producing indirect toxicity.

[0012] This invention also provides an application of traditional Chinese medicine-based carbon nanomaterials in antibacterial applications.

[0013] Furthermore, the herbal-based carbon nanomaterials exhibit antibacterial effects under light irradiation. The material can act as a photosensitizer to generate reactive oxygen species that destroy bacterial structures, and it can also convert light energy into heat energy, leading to bacterial death.

[0014] Furthermore, the light intensity is 3-15 W and the time is 1-3 h, preferably, the light intensity is 13 W and the time is 2 h.

[0015] Furthermore, the bacteria include any one of Escherichia coli, Staphylococcus aureus, and the soft rot pathogen of Chinese cabbage. In this embodiment of the invention, the cell walls of Escherichia coli treated with herbal-based carbon nanomaterials shrink and fuse, resulting in significant morphological changes. As the concentration increases, the survival rate of Escherichia coli gradually decreases, and when the concentration reaches 200 μg / mL, its antibacterial rate is 91%.

[0016] In summary, compared with the prior art, the herbal-based carbon nanomaterials obtained by this invention have excellent photocatalytic antibacterial properties, which can effectively solve the problem of bacterial drug resistance and realize the efficient reuse of herbal resources. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The images show the antibacterial results of the herbal-based carbon nanomaterials in Example 1 of this invention. (a) shows the coating results after treating Escherichia coli with different concentrations of herbal-based carbon nanomaterials in the dark, and (b) shows the coating results after treating Escherichia coli with different concentrations of herbal-based carbon nanomaterials under light.

[0019] Figure 2 The images shown are SEM (scanning electron microscope) images of *E. coli* treated with herbal-based carbon nanomaterials under light in Example 2 of this invention. (a) is an SEM image of the cell membrane of *E. coli* after treatment with PBS (pH 7.4, 20 mM); (b) is an SEM image of the cell membrane of *E. coli* after treatment with NaAc-HAc buffer (pH 5.0, 20 mM); and (c) is an SEM image of the cell membrane of *E. coli* after treatment with NaAc-HAc buffer (pH 5.0, 20 mM) + 100 μg / mL herbal-based carbon nanomaterials. Detailed Implementation

[0020] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0021] Example 1: Preparation of Traditional Chinese Medicine-Based Carbon Nanomaterials Add 1.5g of glutathione and 0.3g of the traditional Chinese medicine *Xiao Yidian Hong* to a reaction vessel, followed by 50 mL of formamide. Stir until the mixture is homogeneous. React at 140-160℃ for 4-8 hours, and then cool to room temperature after the reaction. Centrifuge the resulting reaction solution at 5000 rpm for 10 min to remove the precipitate and collect the supernatant. Dialyze the supernatant using a 3500Da dialysis bag for 5-7 days. After dialysis, collect the liquid in the dialysis bag and concentrate it. Further purification is achieved through repeated freeze-thaw cycles: freeze at -20℃, thaw at room temperature, and filter using a 0.22μm filter membrane. Repeat this process 5-6 times. Finally, freeze-dry the purified sample to obtain the traditional Chinese medicine-based carbon nanomaterial.

[0022] Example 2: Detection of in vitro antibacterial activity of traditional Chinese medicine-based carbon nanomaterials I. Experimental Methods Bacterial culture: A single Escherichia coli ATCC25922 colony from a solid agar plate was inoculated into liquid Luria Bertani (LB) medium and cultured overnight in a shaker at 37°C and 220 rpm. The absorbance (OD600) of the bacterial suspension at 600 nm was then measured using a UV-Vis spectrophotometer. When the OD600 reached 1.0, the bacterial suspension was centrifuged at 3000 rpm for 10 min at 4°C, the supernatant was discarded, and the suspension was washed three times with sterile PBS. After washing, the precipitate was resuspended in sterile PBS and stored in a refrigerator for later use.

[0023] In vitro antibacterial activity assay: A NaAc-HAc buffer solution (pH 5.0, 20 mM) was prepared and sterilized. The bacterial suspension was then serially diluted 100-fold using this solution, and 1 mL was aliquoted into sterile EP tubes. Subsequently, solutions of herbal-based carbon nanomaterials at different concentrations were prepared using sterile water. 10 μL of this solution was added to each EP tube, resulting in final concentrations of 0 (with 10 μL of sterile PBS), 12.5, 25, 50, 100, and 200 μg / mL. After mixing, the solutions were divided into two groups: one group was incubated under 13 W LED light for 2 h, and the other group was incubated in the dark for 2 h to evaluate the antibacterial properties of the herbal-based carbon nanomaterials under different conditions. After incubation, the bacterial suspension was serially diluted 10,000-fold using sterile LB broth. 100 μL of each solution was spread onto plates, with three replicates for each sample. The plates were incubated at 36 ± 1 °C for 24 h, and then photographed and counted.

[0024] II. Experimental Results After 2 hours of LED irradiation, as Figure 1 As shown in ab, the survival rate of Escherichia coli gradually decreased with the increase of the concentration of herbal carbon nanomaterials, showing a concentration-dependent effect. When the concentration of the smaller red carbon dots was 200 μg / mL, its antibacterial rate was 91%.

[0025] Example 3: Morphological characteristics of bacteria treated with herbal-based carbon nanomaterials I. Experimental Methods Morphological characteristics of the bacteria: The bacterial suspension was diluted 100-fold with PBS and NaAc-HAc buffer (pH 5.0, 20 mM). 900 μL of the bacterial suspension was transferred to an EP tube, followed by the addition of 100 μL of herbal-based carbon nanomaterials at a concentration of 1000 μg / mL (PBS + 0 μg / mL herbal-based carbon nanomaterials, NaAc-HAc + 0 μg / mL herbal-based carbon nanomaterials, NaAc-HAc + 100 μg / mL herbal-based carbon nanomaterials). The tubes were incubated under LED (13W) illumination for 2 h. After centrifugation at 3000 rpm for 10 min at 4 °C, the supernatant was discarded, and the precipitate was washed three times with PBS. *E. coli* was fixed with 2.5% glutaraldehyde solution overnight at 4 °C. Subsequently, the bacteria were dehydrated using different concentrations of ethanol solutions (30%, 50%, 70%, 90%, 100%). The final sample was immersed in a clean silicon wafer and dried overnight. After Au was sprayed onto the surface, the sample was imaged using SEM (ZsrissSigma 300).

[0026] II. Experimental Results like Figure 2 The scanning electron microscope (SEM) images of *E. coli* showed that when treated with only PBS and NaAc-HAc buffer and exposed to light for 2 hours, the morphology of *E. coli* remained intact, with clear boundaries and a smooth cell membrane. In contrast, the morphology of *E. coli* treated with NaAc-HAc buffer and carbon dots changed significantly, exhibiting cell wall contraction and fusion. These results indicate that herbal-based carbon nanomaterials possess excellent photocatalytic antibacterial properties.

[0027] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing traditional Chinese medicine-based carbon nanomaterials, characterized in that, The preparation steps include the following: S1: Add glutathione and small red dye to the reaction vessel, then add formamide, stir evenly and then react to obtain the reaction product; S2: After cooling the reaction product obtained in S1 to room temperature, first centrifuge to remove the precipitate, then dialyze the supernatant and collect the liquid in the dialysis bag. S3: The liquid in the dialysis bag is repeatedly frozen and thawed and filtered to achieve concentration and purification. Finally, it is freeze-dried to obtain traditional Chinese medicine-based carbon nanomaterials.

2. The method for preparing traditional Chinese medicine-based carbon nanomaterials according to claim 1, characterized in that, The reaction is carried out at a temperature of 140-160℃ for 4-8 hours.

3. The method for preparing traditional Chinese medicine-based carbon nanomaterials according to claim 1, characterized in that, The dialysis period is 5-7 days.

4. The method for preparing traditional Chinese medicine-based carbon nanomaterials according to claim 1, characterized in that, The centrifugation speed is 3000-10000 r / min, and the time is 10-20 min.

5. A traditional Chinese medicine-based carbon nanomaterial, characterized in that, Prepared by the method according to any one of claims 1-4.

6. The herbal-based carbon nanomaterial according to claim 5, characterized in that, The nanomaterials are smaller than 10 nm in size.

7. The application of the herbal-based carbon nanomaterials according to any one of claims 5-6 in antibacterial applications.

8. The application of the herbal-based carbon nanomaterials according to claim 7 in antibacterial applications, characterized in that, The herbal-based carbon nanomaterials exhibit antibacterial effects under light irradiation.

9. The application of the herbal-based carbon nanomaterials according to claim 8 in antibacterial applications, characterized in that, The light intensity is 3-15 W, and the duration is 1-3 h. Preferably, the light intensity is 13 W, and the duration is 2 h.

10. The application of the herbal-based carbon nanomaterials according to claim 7 in antibacterial applications, characterized in that, The bacteria include any one of Escherichia coli, Staphylococcus aureus, and the pathogen causing soft rot of Chinese cabbage.

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