Preparation method and application of Chinese herbal medicine source carbon dots with pH-responsive photodynamic antibacterial and antioxidant activity
The carbon dots of traditional Chinese medicine prepared by muffle furnace calcination solve the problems of antibiotic resistance and high cost of nanomaterials, and achieve low toxicity, multimodal sterilization and precise treatment effects, making them suitable for the biomedical field.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
- Filing Date
- 2026-01-26
- Publication Date
- 2026-05-12
AI Technical Summary
In existing technologies, the overuse of traditional small molecule antibiotics leads to the emergence of drug-resistant bacteria, the effective components of traditional Chinese medicine are unstable in water, and the preparation of nano-antibacterial materials is complex and costly, making it impossible to precisely target and control the release, resulting in poor antibacterial effects.
Carbon dots derived from traditional Chinese medicine were prepared by muffle furnace calcination. The particles were uniform in size, had good water solubility, and possessed pH-responsive and photodynamic antibacterial capabilities. Precise sterilization was achieved through the synergistic effect of pH response and photodynamics. The preparation process was simplified by combining it with dialysis purification.
The preparation process is simple and low-cost. The carbon dots have good biocompatibility and low cytotoxicity, enabling on-demand treatment and multimodal sterilization, reducing antibiotic resistance, and making them suitable for a variety of applications.
Smart Images
Figure CN122010094A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical nanomaterials technology, and particularly relates to a method for preparing carbon dots derived from traditional Chinese medicine with pH-responsive photodynamic antibacterial and antioxidant activities and their applications. Background Technology
[0002] Bacterial infections pose a significant threat to human health, and antibiotics remain the primary means of combating them. However, the overuse of traditional small-molecule antibiotics and bacterial evolution have led to the emergence of numerous drug-resistant bacteria. These resistant bacteria, especially those with cross-resistance, pose a serious threat to human health. Furthermore, the preparation of traditional nanomaterials for antibacterial purposes suffers from several problems: some preparation methods involve complex processes and demanding reaction conditions (such as high temperature and pressure, and precious metal catalysis), resulting in high production costs; low solubility; and unstable active ingredients. This prevents the effective antibacterial components in traditional Chinese medicine from existing in a large, stable form in water, and also hinders the precise targeting of the infection site and control of the release rate.
[0003] Therefore, the development of novel drugs with highly efficient antibacterial activity is urgently needed. In recent years, various carbon-based nanomaterials, such as carbon dots, have become promising new nano-antibacterial agents due to their unique physicochemical properties, such as nanoscale size, high stability, good water solubility, and biocompatibility. Summary of the Invention
[0004] The purpose of this invention is to provide a method for preparing carbon dots derived from traditional Chinese medicine with pH-responsive photodynamic antibacterial and antioxidant activities and their application. The carbon dots derived from traditional Chinese medicine have a uniform particle size distribution (~2nm) and good water solubility. They also have low cytotoxicity (cell viability ≥95% at a concentration of 200μg / mL) and good biocompatibility, thus providing a guarantee for their application in the biomedical field.
[0005] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, the present invention provides a method for preparing carbon dots derived from traditional Chinese medicine, comprising the following steps: Step 1: Weigh rhubarb and soak it in a 20% ethanol solution to obtain rhubarb extract; Step 2: After sonicating the rhubarb extract from Step 1 for 10-15 minutes, take an appropriate amount of the rhubarb extract and add it to a ceramic boat. Place the boat in a muffle furnace and calcine at 260-280℃ for 60-90 minutes to obtain a black solid. Step 3: Dissolve the black solid obtained in Step 2 in deionized water and sonicate for 10-15 minutes. Centrifuge to obtain the supernatant, and put the obtained supernatant into a dialysis bag for dialysis to obtain the carbon dots from the Chinese herbal medicine.
[0006] In the above technical solution, in step 1, the percentage of rhubarb to ethanol solution is: 10g: (100-150)mL.
[0007] In the above technical solution, in step 3, the cutoff molecular weight of the dialysis bag is 10000 Da.
[0008] Secondly, the present invention provides a carbon dot derived from traditional Chinese medicine, which is prepared by the above-described preparation method.
[0009] Thirdly, the present invention provides the application of the above-mentioned carbon dots from traditional Chinese medicine sources in antibacterial applications.
[0010] In the above technical solutions, the antibacterial effect is manifested in the resistance to Staphylococcus aureus.
[0011] Fourthly, the present invention provides the application of the above-mentioned carbon dots from traditional Chinese medicine sources in antioxidant properties.
[0012] Fifthly, this invention provides the application of the above-mentioned carbon dots from traditional Chinese medicine sources in the detection of heavy metal ions and small biological molecules.
[0013] Sixthly, the present invention provides the application of the above-mentioned carbon dots from traditional Chinese medicine sources in monitoring the intracellular microenvironment.
[0014] In a seventh aspect, the present invention provides the application of the above-mentioned carbon dots derived from traditional Chinese medicine in the field of fluorescence imaging.
[0015] The beneficial effects of this invention are as follows: 1. The preparation process of this invention is simple, using muffle furnace calcination, which does not require complex equipment or subsequent modification steps. The reaction conditions are mild, the cost is low, and it is easy to scale up production.
[0016] 2. The carbon dots derived from Chinese herbal medicines in this invention possess both pH and photodynamic responses, enabling them to perform different functions according to actual needs and making them suitable for various application scenarios.
[0017] 3. The carbon dots derived from Chinese herbal medicines in this invention have good biocompatibility, low toxicity, and excellent water solubility. Compared with the raw material rhubarb, the carbon dots reduce the cytotoxicity of rhubarb and improve the water solubility of the effective antibacterial components in rhubarb. Attached Figure Description
[0018] Figure 1 This is a flowchart of the preparation method of the present invention.
[0019] Figure 2 This is a schematic diagram of plate antibacterial methods using calcination and hydrothermal methods under pH 5.5 conditions.
[0020] Figure 3 This is a schematic diagram of plate antibacterial treatment using calcination and hydrothermal methods under pH 7.4 conditions.
[0021] Figure 4 These are images of a DPPH scavenging experiment under near-neutral conditions (pH 7.4) (the concentration of rhubarb carbon spots gradually increases from left to right).
[0022] Figure 5 These are images of an ABTS free radical scavenging experiment under near-neutral conditions (pH 7.4) (the concentration of rhubarb carbon dots gradually increases from left to right). Detailed Implementation
[0023] 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.
[0024] This invention provides a method for preparing carbon dots derived from traditional Chinese medicine with pH-responsive photodynamic antibacterial and antioxidant activities, and their applications, such as... Figure 1 As shown, the preparation method includes three steps: solution leaching, muffle furnace calcination, and carbon dot purification. This method is simple, safe to operate, and requires no large instruments or high-pressure environment. The prepared carbon dots derived from traditional Chinese medicine achieve pH response through protonation / deprotonation, while inheriting the potential photosensitivity of the original drug. Under specific pH and visible light excitation, their antibacterial efficiency can be greatly improved, showing promising application prospects in multiple fields such as antibacterial, detection, and fluorescence imaging.
[0025] In the field of antibacterial agents, the core advantage of the carbon dots in this invention lies in their ability to achieve on-demand treatment and multimodal sterilization, reducing the development of antibiotic resistance. Specifically: (1) Synergistic effect of photodynamic therapy (PDT) and photothermal therapy (PTT) Principle: The carbon dots of this invention act as photosensitizers, generating reactive oxygen species (ROS, such as singlet oxygen) under light of a specific wavelength, which disrupt the bacterial cell membrane (PDT). Simultaneously, some carbon dots possess excellent photothermal conversion efficiency, converting light energy into heat energy to "burn" bacteria (PTT).
[0026] (2) Enhanced pH response: The bacterial infection microenvironment is usually weakly acidic. Carbon dots with pH response can undergo charge reversal (e.g., from negative to positive charge) in this acidic environment, making them more easily adsorbed by the negatively charged bacterial surface, thus killing bacteria more precisely and efficiently under light, while having extremely low toxicity to healthy cells under normal physiological pH (neutral).
[0027] In the field of detection, the carbon dots of this invention primarily utilize changes in fluorescence intensity or color to detect specific ions, molecules, or environmental parameters. Specifically: (1) Detection of heavy metal ions and small biological molecules Principle: Certain metal ions (such as Fe) 2+ Cu 2+ Biomolecules (such as glutathione and glucose) may quench the fluorescence of carbon dots, or react with carbon dots under specific pH conditions to restore fluorescence ("switching" effect).
[0028] (2) Monitoring of the intracellular microenvironment Due to its pH sensitivity, the carbon dots of this invention are often used as probes to detect the state of lysosomes (acidic environment) or mitochondria in cells, and even to distinguish between cancer cells (which are usually metabolically active and have an acidic environment) and normal cells.
[0029] In the field of fluorescence imaging, the excellent optical properties and biocompatibility of carbon dots are utilized to achieve high-resolution, high-contrast biological imaging. Specifically, this manifests as follows: (1) Tumor-targeted imaging Principle: The tumor microenvironment is usually weakly acidic (Warburg effect).
[0030] Application: pH-responsive carbon dots are designed to fluoresce weakly or "off" in the bloodstream (neutral pH), but immediately "on" or enhance fluorescence once they reach the tumor site (acidic pH). This "activatable" imaging modality greatly improves the signal-to-noise ratio, aiding in the precise localization of early-stage tumors.
[0031] (2) Integrated diagnosis and treatment Combination: Integrating imaging and treatment. Carbon dots are first used to locate lesions (such as sites of bacterial infection or tumors) using fluorescence imaging. After confirming the location, their photosensitivity is utilized to apply light for PDT / PTT treatment. The entire process is visualized, allowing for real-time monitoring of treatment effectiveness.
[0032] The carbon dots derived from traditional Chinese medicine prepared in this invention have a uniform particle size distribution (~2 nm) and good water solubility. Furthermore, these carbon dots exhibit low cytotoxicity (cell viability ≥95% at a concentration of 200 μg / mL) and good biocompatibility, providing a guarantee for their application in the biomedical field.
[0033] The carbon dots derived from traditional Chinese medicine in this invention can be used for antibacterial purposes. The bacteria is Staphylococcus aureus.
[0034] Specifically, the antibacterial behavior is as follows: the carbon dots derived from this traditional Chinese medicine exhibit excellent antibacterial ability only under conditions of weak acidity (pH 5.5-6.5) and visible white light illumination. Under conditions of weak acidity and neutrality without light, as well as under neutrality (pH 7-7.4) and visible white light illumination, the antibacterial ability of the carbon dots derived from this traditional Chinese medicine is greatly weakened.
[0035] Antibacterial mechanism: Under weakly acidic conditions (pH 5.5-6.5) and activated by visible white light, the carbon dots derived from this traditional Chinese medicine can absorb photons and transition from the ground state to the excited singlet state, transferring energy to oxygen molecules. The oxygen molecules absorb energy and undergo electron spin transitions, producing a large amount of singlet oxygen. 1 O2 directly kills bacteria; after turning off the light... 1 O2 generation stops. This can be achieved by adjusting pH and light conditions to turn it on / off. 1 The generation of O2.
[0036] Meanwhile, the carbon dots derived from traditional Chinese medicine in this invention can be used for antioxidant applications.
[0037] Specifically, carbon dots exhibit good antioxidant capacity under neutral conditions (pH 7-7.4), but their antioxidant capacity is greatly weakened under weakly acidic conditions (pH 5.5-6.5).
[0038] Example 1 This embodiment provides a method for preparing carbon dots derived from traditional Chinese medicine with pH-responsive photodynamic antibacterial and antioxidant activities, including the following steps: (1) Weigh 10g of rhubarb and soak it in 100mL of 20% ethanol solution for 24h to obtain rhubarb extract.
[0039] (2) After sonicating the rhubarb extract in (1) for 10 min, take an appropriate amount of the rhubarb extract and add it to a ceramic boat. Place it in a muffle furnace at 260℃ for 90 min to obtain a black solid.
[0040] (3) Dissolve the black solid obtained in (2) in deionized water and sonicate for 15 min. Centrifuge to obtain the supernatant and put the obtained supernatant into a dialysis bag (with a molecular weight of 10000 Da) for dialysis for 48 h.
[0041] Example 2 This embodiment provides a method for preparing carbon dots derived from traditional Chinese medicine with pH-responsive photodynamic antibacterial and antioxidant activities, including the following steps: (1) Weigh 10g of rhubarb and soak it in 100mL of 20% ethanol solution for 24h to obtain rhubarb extract.
[0042] (2) After sonicating the rhubarb extract in (1) for 10 min, take an appropriate amount of the rhubarb extract and add it to a ceramic boat. Place it in a muffle furnace at 260℃ for 60 min to obtain a black solid.
[0043] (3) Dissolve the black solid obtained in (2) in deionized water and sonicate for 15 min. Centrifuge to obtain the supernatant and put the obtained supernatant into a dialysis bag (with a molecular weight of 10000 Da) for dialysis for 48 h.
[0044] Example 3 This embodiment provides a method for preparing carbon dots derived from traditional Chinese medicine with pH-responsive photodynamic antibacterial and antioxidant activities, including the following steps: (1) Weigh 10g of rhubarb and soak it in 100mL of 20% ethanol solution for 24h to obtain rhubarb extract.
[0045] (2) After sonicating the rhubarb extract in (1) for 10 min, take an appropriate amount of the rhubarb extract and add it to a ceramic boat. Place it in a muffle furnace at 280℃ for 90 min to obtain a black solid.
[0046] (3) Dissolve the black solid obtained in (2) in deionized water and sonicate for 15 min. Centrifuge to obtain the supernatant and put the obtained supernatant into a dialysis bag (with a molecular weight of 10000 Da) for dialysis for 48 h.
[0047] Example 4 This embodiment provides a method for preparing carbon dots derived from traditional Chinese medicine with pH-responsive photodynamic antibacterial and antioxidant activities, including the following steps: (1) Weigh 10g of rhubarb and soak it in 100mL of 20% ethanol solution for 24h to obtain rhubarb extract.
[0048] (2) After sonicating the rhubarb extract in (1) for 10 min, take an appropriate amount of the rhubarb extract and add it to a ceramic boat. Place it in a muffle furnace at 260℃ for 80 min to obtain a black solid.
[0049] (3) Dissolve the black solid obtained in (2) in deionized water and sonicate for 15 min. Centrifuge to obtain the supernatant and put the obtained supernatant into a dialysis bag (with a molecular weight of 10000 Da) for dialysis for 48 h.
[0050] Example 5 This embodiment provides a method for preparing carbon dots derived from traditional Chinese medicine with pH-responsive photodynamic antibacterial and antioxidant activities, including the following steps: (1) Weigh 10g of rhubarb and soak it in 150mL of 20% ethanol solution for 24h to obtain rhubarb extract.
[0051] (2) After sonicating the rhubarb extract in (1) for 10 min, take an appropriate amount of the rhubarb extract and add it to a ceramic boat. Place it in a muffle furnace at 260℃ for 90 min to obtain a black solid.
[0052] (3) Dissolve the black solid obtained in (2) in deionized water and sonicate for 15 min. Centrifuge to obtain the supernatant and put the obtained supernatant into a dialysis bag (with a molecular weight of 10000 Da) for dialysis for 48 h.
[0053] Example 6 This embodiment provides a method for preparing carbon dots derived from traditional Chinese medicine with pH-responsive photodynamic antibacterial and antioxidant activities, including the following steps: (1) Weigh 10g of rhubarb and soak it in 100mL of 20% ethanol solution for 24h to obtain rhubarb extract.
[0054] (2) After sonicating the rhubarb extract in (1) for 15 minutes, take an appropriate amount of the rhubarb extract and add it to a ceramic boat. Place it in a muffle furnace at 260°C for 90 minutes to obtain a black solid.
[0055] (3) Dissolve the black solid obtained in (2) in deionized water and sonicate for 15 min. Centrifuge to obtain the supernatant and put the obtained supernatant into a dialysis bag (with a molecular weight of 10000 Da) for dialysis for 48 h.
[0056] Experimental process Rhubarb carbon dots were prepared using the calcination method described in Example 1. Simultaneously, rhubarb carbon dots were prepared using a hydrothermal method. The specific steps of the hydrothermal method were as follows: 10 ml of rhubarb extract was transferred to a 25 ml stainless steel autoclave lined with polytetrafluoroethylene, and reacted at 180°C for 8 hours. After cooling to room temperature, the mixture was centrifuged three times at 9000 rpm for 10 min, and the supernatant was collected. The supernatant was then dialyzed at 10000 Da for 48 hours.
[0057] Plate antibacterial test: Staphylococcus aureus culture was incubated overnight at 37°C, then diluted to 1×10⁻⁶ with phosphate buffer (pH 5.5, 10 mM). 6 ~1×10 7 CFU was incubated with a specific concentration of rhubarb carbon dots for 15 min, and then irradiated under an LED lamp for 30 min. 10 μL of the above solution was diluted with phosphate buffer (pH 5.5, 10 mM) to 500 μL, and 50 μL of the diluted solution was plated on LB agar plates and incubated at 37 °C for 18 h. The colony counts on each plate were statistically analyzed.
[0058] like Figure 2 and 3As shown, the plate antibacterial method proves that carbon dots prepared by calcination can kill bacteria under slightly acidic conditions and light irradiation for 30 minutes. Carbon dots prepared by hydrothermal method have a lower inhibitory efficiency on bacteria under the same conditions than those prepared by calcination.
[0059] ABTS free radical scavenging experiment: 38.4 mg ABTS and 6.59 mg K2S2O8 were dissolved in 10 mL of water and stored in the dark for more than 12 hours to generate ABTS. •+ The solution was then diluted until the absorbance at 734 nm was between 0.6 and 0.9. Then, a specific concentration of rhubarb carbon dots (experimental group) or phosphate buffer at different pH values (control group) was added. After standing for 15 minutes, the absorbance of the mixed solution at 734 nm was measured using a microplate reader.
[0060] DPPH removal experiment: 100 μM DPPH of rhubarb carbon dots (experimental group) and blank control group (control group) were incubated in ethanol in the dark for 1 h, and then the absorbance of the mixed solution at 517 nm was measured.
[0061] like Figure 4 and 5 As shown, the antioxidant capacity of carbon dots was verified by the ABTS and DPPH methods. Under neutral conditions, carbon dots could cause the green color of ABTS to fade and the purple color of DPPH to fade.
[0062] 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 carbon dots derived from traditional Chinese medicine, characterized in that: Includes the following steps: Step 1: Weigh rhubarb and soak it in a 20% ethanol solution to obtain rhubarb extract; Step 2: After ultrasonically treating the rhubarb extract from Step 1 for 10-15 minutes, take an appropriate amount of the rhubarb extract and add it to a ceramic boat. Place the boat in a muffle furnace and calcine at 260-280℃ for 60-90 minutes to obtain a black solid. Step 3: Dissolve the black solid obtained in Step 2 in deionized water and sonicate for 10-15 minutes. Centrifuge to obtain the supernatant, and put the obtained supernatant into a dialysis bag for dialysis to obtain the carbon dots from the Chinese herbal medicine.
2. The preparation method according to claim 1, characterized in that: In step 1, the percentage of rhubarb to ethanol solution is: 10g: (100-150)mL.
3. The preparation method according to claim 1, characterized in that: In step 3, the cutoff molecular weight of the dialysis bag is 10,000 Da.
4. A carbon dot derived from traditional Chinese medicine, characterized in that: It is prepared by the preparation method described in any one of claims 1-3.
5. The application of the carbon dots derived from traditional Chinese medicine as described in claim 4 in antibacterial applications.
6. The application according to claim 5, characterized in that: Its antibacterial properties are manifested in its resistance to Staphylococcus aureus.
7. The application of the carbon dots derived from traditional Chinese medicine as described in claim 4 in terms of antioxidant properties.
8. The application of the carbon dots derived from traditional Chinese medicine as described in claim 4 in the detection of heavy metal ions and small biological molecules.
9. The application of the carbon dots derived from traditional Chinese medicine as described in claim 4 in monitoring the intracellular microenvironment.
10. The application of the carbon dots derived from traditional Chinese medicine as described in claim 4 in the field of fluorescence imaging.