Traditional Chinese medicine compound carbon dots for treating granulomatous mastitis and preparation method and application thereof

Abstract

The application discloses a traditional Chinese medicine compound carbon dot for treating granulomatous mastitis and a preparation method and application thereof, and relates to the technical field of traditional Chinese medicine carbon dots. The raw materials for preparing the carbon dot include, by weight, 30 parts of Astragalus membranaceus, 15 parts of Atractylodes lancea, 30 parts of Smilax glabra, 10 parts of Alisma orientale, 10 parts of Honeysuckle and 10 parts of Forsythia. The preparation method comprises the following steps: mixing the raw materials, high-temperature calcining, grinding into powder, boiling extraction with deionized water, and finally filtering through a microporous filter membrane and purifying through dialysis. The traditional Chinese medicine compound carbon dot has a nanoscale size and excellent water solubility, overcomes the defects of traditional Chinese medicine, such as oral administration only and unstable drug efficacy, and realizes precise local injection administration to breast lesions. The carbon dot has high biocompatibility, can effectively reduce the pathological structure of granuloma, and significantly down-regulates inflammatory factors such as IL-6, IL-17 and TNF-alpha, thereby providing a safe and efficient solution for the treatment of granulomatous mastitis.

Description

Technical Field

[0001] This application belongs to the field of carbon dot technology of traditional Chinese medicine, and particularly relates to carbon dots of traditional Chinese medicine compound for the treatment of granulomatous mastitis, as well as their preparation methods and applications. Background Technology

[0002] Granulomatous lobular mastitis (GLM) is a special type of inflammatory breast disease characterized by the formation of non-caseous necrotic granulomas within the breast lobules, accounting for approximately 24% of all inflammatory breast diseases. GLM lesions are extensive, have a prolonged course, are prone to forming fistulas or sinuses, and are often slow to heal, causing significant damage to the appearance of the breast and severely impacting the patient's mental health and quality of life.

[0003] Currently, the main treatments for granulomatous mastitis include hormone therapy, immunosuppressants, local surgery, and traditional Chinese medicine. However, all existing treatments have significant limitations. On the one hand, Western medicine treatments suffer from long treatment cycles, numerous adverse reactions, severe damage to breast appearance, and a high recurrence rate, leading to persistent and difficult-to-cure granulomatous mastitis. On the other hand, with the deepening understanding of this disease in traditional Chinese medicine, TCM, with its multi-component, multi-pathway, and multi-target regulatory characteristics, has shown broad therapeutic prospects and unique advantages in relieving symptoms, regulating immunity, and reducing recurrence rates.

[0004] However, traditional Chinese medicine requires long treatment cycles, leading to low patient compliance. More importantly, the active ingredients in traditional Chinese medicinal materials generally have large molecular weights and poor water solubility, resulting in low bioavailability when directly decocted and taken, thus limiting their clinical application. Summary of the Invention

[0005] The purpose of this invention is to provide a traditional Chinese medicine compound carbon dot for treating granulomatous mastitis, its preparation method and application, aiming to solve the technical problems of poor water solubility and low bioavailability of existing traditional Chinese medicines, especially the fact that traditional Chinese medicines can only be taken orally and cannot be injected locally into the lesion.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, embodiments of this application provide a traditional Chinese medicine compound carbon dot for treating granulomatous mastitis, wherein the raw materials for preparing the traditional Chinese medicine compound carbon dot include, by weight: Astragalus membranaceus 30 parts, Curcuma zedoaria (processed with vinegar) 15 parts, Smilax glabra 30 parts, Alisma plantago-aquatica 10 parts, Lonicera japonica 10 parts, Forsythia suspensa 10 parts.

[0007] Secondly, this application also provides a method for preparing the carbon dots of a traditional Chinese medicine compound for treating granulomatous mastitis as described above, comprising the following steps: Step 1, Mixing and Calcination: The Astragalus membranaceus, Curcuma zedoaria, Smilax glabra, Alisma plantago-aquatica, Lonicera japonica and Forsythia suspensa weighed according to the above weight proportions are mixed and placed in a crucible and sealed. Then, they are calcined in a muffle furnace at 350°C. After cooling, the medicine in the crucible is ground into powder. Step 2, Boiling in a water bath: Mix the obtained powder with deionized water and boil it in a water bath; Step 3, Filtration and Dialysis: The extract is filtered to remove residues, the solution is concentrated and then dialyzed to obtain the carbon dots of the Chinese herbal compound.

[0008] Optionally, in step one, the crucible is covered with aluminum foil to form a seal.

[0009] Optionally, in step two, the powder is mixed with 3 liters of deionized water and boiled twice in a water bath, each time for 1 hour.

[0010] Optionally, in step three, a filter membrane with a pore size of 0.22 μm is used for filtration.

[0011] Optionally, in step three, a dialysis bag with a molecular weight cutoff of 1 kDa is used and the dialysis is performed in deionized water for 168 hours.

[0012] Thirdly, the embodiments of this application also provide the application of the above-mentioned compound carbon dots of traditional Chinese medicine or the compound carbon dots of traditional Chinese medicine obtained by the above preparation method in the preparation of drugs for treating granulomatous mastitis.

[0013] Optionally, the drug is an aqueous solution and is used for local injection into granulomatous mastitis lesions.

[0014] Compared with the prior art, the beneficial effects of the embodiments of this application are: (1) This invention prepares a compound traditional Chinese medicine carbon dot from a combination of Astragalus membranaceus, Curcuma zedoaria (processed with vinegar), Smilax glabra, Alisma plantago-aquatica, Lonicera japonica, and Forsythia suspensa, overcoming the shortcomings of poor water solubility and low oral bioavailability of large molecular active ingredients in traditional Chinese medicine. With the nanoscale size of less than 10 nm and high water solubility of the carbon dot, it can more effectively cross biological barriers and overcome the shortcomings of traditional Chinese medicine that can only be taken orally and has unstable efficacy. It realizes precise local injection of drugs to granulomatous mastitis lesions, improving the stability, convenience and effective targeted delivery rate of local drug administration.

[0015] (2) The carbon dots of the traditional Chinese medicine compound of the present invention not only retain the advantages of the original compound in multi-component and multi-target regulation, but also enhance its pharmacological activity through nanotechnology. Animal experimental pathological observation results confirm that after intervention with the carbon dots of the traditional Chinese medicine compound of the present invention, the granuloma pathological structure of rat mammary tissue is significantly reduced, which can reduce the infiltration of inflammatory cells such as lymphocytes and plasma cells, and reduce the proliferation of epithelial cells, and has a repairing effect on damaged mammary tissue.

[0016] (3) ELISA results showed that the traditional Chinese medicine compound carbon dots of the present invention can effectively reverse the abnormal inflammatory markers caused by granulomatous mastitis. The traditional Chinese medicine compound carbon dots group can reduce the expression levels of IL-6 and TNF-α in the serum of model rats. Its downregulation effect on IL-6 is comparable to that of the positive drug (triamcinolone acetonide), and it can also significantly reduce the level of IL-17, showing good anti-inflammatory efficacy.

[0017] (4) The carbon dots of the traditional Chinese medicine compound of the present invention inherit the characteristics of natural carbon sources, and reduce potential systemic toxic side effects while retaining the efficacy. CCK8 cytotoxicity test showed that the carbon dots of the traditional Chinese medicine compound of the present invention had no obvious toxicity to normal breast epithelial cells MCF-10a in the concentration range of 0.2 mg / ml, 0.4 mg / ml, 0.8 mg / ml, 1.2 mg / ml, 1.6 mg / ml and 2.0 mg / ml, demonstrating good in vitro and in vivo biocompatibility and high safety for clinical application.

[0018] (5) The preparation method of the present invention uses traditional Chinese medicinal materials or residues as carbon sources to prepare carbon dots through a hydrothermal method, turning waste into treasure and realizing the high-value utilization of Chinese medicinal materials. Compared with the complex nanomedicine synthesis process, this preparation method has the characteristics of simple operation and low cost, providing a new idea for green and sustainable development and having significant potential for large-scale industrial production. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The image shows the fluorescence effect of carbon dots in a traditional Chinese medicine compound.

[0021] Figure 2 The image shows electron microscopy images of carbon dots in traditional Chinese medicine compound preparations, including their size.

[0022] Figure 3 The XPS full spectrum of carbon dots in the traditional Chinese medicine compound is shown.

[0023] Figure 4 The fine XPS C1s spectrum of carbon dots of the traditional Chinese medicine compound is shown.

[0024] Figure 5 The fine XPS N 1s spectrum of carbon dots of the traditional Chinese medicine compound is shown.

[0025] Figure 6 The fine XPS O 1s spectrum of carbon dots in the traditional Chinese medicine compound is shown.

[0026] Figure 7 The XRD diffraction pattern of carbon dots in a traditional Chinese medicine compound is shown.

[0027] Figure 8 The infrared spectrum of carbon dots in a traditional Chinese medicine compound is shown.

[0028] Figure 9 The ultraviolet spectrum of carbon dots in a traditional Chinese medicine compound is shown.

[0029] Figure 10 The fluorescence spectrum of carbon dots in a traditional Chinese medicine compound is shown.

[0030] Figure 11 The image shows the CCK8 cytotoxicity test results of carbon dots in a traditional Chinese medicine compound.

[0031] Figure 12 The pathological H&E staining images of rat mammary tissues in each group are shown, where A is the blank group, B is the model group, C is the compound carbon dot group, and D is the positive control group.

[0032] Figure 13 The ELISA results of relevant inflammatory factors in the serum of rats in each group are shown. I represents the change in prolactin (PRL) level, II represents the change in interleukin-6 (IL-6) level, III represents the change in interleukin-17 (IL-17) level, IV represents the change in transforming growth factor-β (TGF-β) level, and V represents the change in tumor necrosis factor-α (TNF-α) level. Detailed Implementation

[0033] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of this application.

[0034] It should be noted that when a component is referred to as being "fixed to" or "set on" another component, it can be directly on or indirectly on that other component. When a component is referred to as being "connected to" another component, it can be directly connected to or indirectly connected to that other component.

[0035] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0036] To illustrate the technical solution described in this application, specific embodiments are provided below.

[0037] Example 1: Preparation of carbon dots for a traditional Chinese medicine compound used to treat granulomatous mastitis This embodiment provides a traditional Chinese medicine compound carbon dot for treating granulomatous mastitis, and its specific preparation steps are as follows: (1) Weighing and mixing raw materials: Weigh each Chinese herbal raw material by weight, specifically: Astragalus membranaceus 30 g, Curcuma zedoaria 15 g, Smilax glabra 30 g, Alisma plantago-aquatica 10 g, Lonicera japonica 10 g, Forsythia suspensa 10 g, and mix the above compound Chinese herbs thoroughly.

[0038] (2) High-temperature calcination: Place the mixed Chinese medicine into a crucible and cover the top of the crucible with aluminum foil to form a sealed environment. Then, place the sealed crucible in a muffle furnace and calcine at 350°C (the calcination time can be set according to the actual process, for example, 1 hour). After calcination, allow it to cool and grind the medicine in the crucible into powder.

[0039] (3) Water bath boiling extraction: Mix the obtained drug powder with 3 L of deionized water evenly and place it in a water bath for boiling extraction. Boil extraction is performed twice, with each boiling time being 1 hour.

[0040] (4) Filtration and dialysis: After extraction, the solution was filtered using a 0.22 μm pore size filter membrane to remove residues. The filtered solution was concentrated and then placed in a dialysis bag with a molecular weight cutoff of 1 kDa. It was dialyzed continuously in deionized water for 168 hours to finally obtain the carbon dot aqueous solution of the traditional Chinese medicine compound.

[0041] Experimental Example 1: Physicochemical Characterization of Carbon Dots in Traditional Chinese Medicine Compound Formulas To verify the structure and physicochemical properties of the product prepared in Example 1, a series of characterization tests were performed, including detecting carbon dot size, ultraviolet light, infrared spectroscopy, XRD, XPS, and fluorescence, confirming the successful preparation of carbon dots from the traditional Chinese medicine compound. The results are as follows: 1. Fluorescence visual observation and morphology detection Please see Figure 1 As shown, Figure 1 The image shows the fluorescence effect of carbon dots in a traditional Chinese medicine compound. Figure 1As can be seen, under ultraviolet light irradiation, the carbon dot aqueous solution of the traditional Chinese medicine compound of the present invention (left side) emits obvious fluorescence, while the pure water of the control group (right side) shows no fluorescence.

[0042] Please see Figure 2 As shown, Figure 2 The image shows the size of the carbon dots obtained from the traditional Chinese medicine compound under an electron microscope (EMS) at a scale bar of 20 nm. EM analysis revealed that the prepared carbon dots had regular morphology and a size less than 10 nm.

[0043] 2. X-ray photoelectron spectroscopy (XPS) analysis The surface composition and electronic states of carbon dots in traditional Chinese medicine compound preparations were characterized using X-ray photoelectron spectroscopy (XPS).

[0044] Please see Figures 3 to 6 As shown, Figures 3 to 6 The XPS full spectrum and fine spectrum of carbon dots in traditional Chinese medicine compound preparations are shown. Figure 3 The full spectrum shown indicates that the carbon point is primarily composed of three elements: carbon (C), nitrogen (N), and oxygen (O). The horizontal axis represents the binding energy, measured in electron volts (eV); the vertical axis represents the photoelectron signal intensity, measured in au (any unit).

[0045] Depend on Figure 4 The fine C 1s spectrum shown indicates peaks at 284.6 eV, 285.9 eV, and 287.6 eV, corresponding to the CC / CN bond, CO bond, and C=O bond, respectively.

[0046] exist Figure 5 In the N 1s fine spectrum shown, the peak at 399.6 eV corresponds to the CN bond, and the peak at 401.1 eV corresponds to the NH bond.

[0047] exist Figure 6 In the O 1s fine spectrum shown, the peaks at 532.7 eV and 531.6 eV are attributed to CO and C=O bonds, respectively.

[0048] 3. X-ray diffraction (XRD) analysis Please see Figure 7 As shown, Figure 7 The XRD diffraction pattern of carbon dots in a traditional Chinese medicine compound is shown. The horizontal axis represents the diffraction angle 2θ in degrees (°); the vertical axis represents the relative intensity of the diffraction peaks in au (arbitrary units).

[0049] The XRD pattern shows peaks at 28.9°, 35.5°, 38.9°, 42.7°, 47.1°, and 48.0°. The 28.9° peak is the dominant peak with a high intensity, corresponding to the (002) crystal plane of the carbon material, with an interplanar spacing of d = 0.31 nm. This spacing is slightly lower than the ideal graphite (002) crystal plane spacing of d = 0.335 nm, indicating that the carbon quantum dots in the traditional Chinese medicine compound have a low-crystallinity graphite-like structure with relatively tight interlayer stacking and still exhibiting disordered defects, representing the characteristic crystal plane signal of carbon skeleton sp2 hybridization. The other high-angle secondary peaks correspond to distortion peaks of the (100) and (101) crystal planes. Due to the nano-sized carbon quantum dots, the crystal plane signal is weakened, and the peak positions shift. Overall, the main peak is broadened, and the secondary peaks are weaker, indicating that the carbon quantum dots have low crystallinity and a large number of surface active sites (corresponding to the abundant oxygen-containing functional groups on the surface characterized by infrared spectroscopy), which is beneficial for applications such as adsorption and catalysis.

[0050] 4. Infrared spectroscopy analysis Please see Figure 8 As shown, Figure 8 The infrared spectrum of carbon dots in a traditional Chinese medicine compound is shown. Figure 8 The presence of abundant oxygen-containing functional groups on the surface of the carbon dots was confirmed. 3387 cm -1 The peak at the position corresponds to the absorption peak caused by the stretching vibration of the hydroxyl group. The peak intensity is relatively large, indicating a high content.

[0051] 2929 cm -1 The peak at the position is due to the absorption peak caused by the asymmetric stretching vibration of the CH bond in the methyl or methylene group.

[0052] 1623 cm -1 The high-intensity absorption peak at the position is caused by the symmetrical stretching vibration of C=C bond or the stretching vibration of C=O bond, and the content is relatively high.

[0053] 1389 cm -1 The peak at this position is an absorption peak caused by the stretching vibration of the CN bond.

[0054] 1032 cm -1 The peak at position is an absorption peak caused by the stretching vibration of the CO bond.

[0055] 574 cm -1 The peak at the position is an absorption peak caused by the bending vibration of the OH bond in the hydroxyl group.

[0056] The horizontal axis represents the wave number, measured in cm. -1 (inverted centimeters); the vertical axis represents the infrared absorption intensity, in au (any unit).

[0057] 5. Ultraviolet and fluorescence spectroscopy analysis Please see Figure 9 As shown, Figure 9 The ultraviolet spectrum of carbon dots in a traditional Chinese medicine compound is shown. Figure 9 It can be seen that the peak at 294 nm corresponds to the abundant aromatic rings in the compound raw materials of this invention. → The electronic transition; the peak at 338 nm corresponds to the lone pair of electrons in the oxygen-containing hydroxyl group of carbon quantum dots, CO, or C=O. The orbital transition process, i.e., n → The transition process. The horizontal axis represents wavelength (nm); the vertical axis represents absorption intensity (au, any unit).

[0058] Please see Figure 10 As shown, Figure 10 The fluorescence spectrum of carbon dots in a traditional Chinese medicine compound is shown. Figure 10 It can be seen that the excitation peak at 495 nm is due to the n→ α-axis of oxygen-containing functional groups such as hydroxyl groups, C=O, and CO bonds on the surface of carbon quantum dots. The photon wavelength at this wavelength efficiently excites electrons to transition from the ground state to the excited state, making it the optimal excitation wavelength for inducing 650 nm fluorescence emission. 650 nm is attributed to fluorescence emission from electrons transitioning from the surface state back to the ground state. Peak intensity is positively correlated with the density of surface functional groups and the degree of defects. A larger peak intensity indicates a greater variety of functional groups and abundant surface defects. The horizontal axis represents wavelength in nanometers (nm), and the vertical axis represents the count in au (arbitrary units).

[0059] Experimental Example 2: In vitro cytotoxicity evaluation of carbon dots in traditional Chinese medicine compound preparations This experiment uses the CCK8 assay to detect the cytotoxicity of the prepared traditional Chinese medicine compound carbon dots.

[0060] Experimental Methods: Cells were seeded at a density of 8000 MCF-10a normal mammary epithelial cells per well in 96-well plates. After incubation for 24 hours, the original culture medium in each well was discarded, and different concentrations (0.2 mg / ml, 0.4 mg / ml, 0.8 mg / ml, 1.2 mg / ml, 1.6 mg / ml, 2.0 mg / ml) of a traditional Chinese medicine compound carbon dot aqueous solution diluted with complete culture medium were added. The cells were then incubated for another 24 hours. Cells were washed with phosphate-buffered saline to remove excess carbon dots and culture medium. Subsequently, 10 μL of CCK-8 solution was mixed with 100 μL of culture medium and added to each well, and incubated for 2 h. Cell viability was detected by measuring absorbance (OD value) at 450 nm using a microplate reader.

[0061] Experimental results: Please see Figure 11 As shown, Figure 11 The image shows the CCK8 cytotoxicity detection results of carbon dots in a traditional Chinese medicine compound. Figure 11 As can be seen, within the concentration ranges of 0.2 mg / ml, 0.4 mg / ml, 0.8 mg / ml, 1.2 mg / ml, 1.6 mg / ml, and 2.0 mg / ml, the relative cell viability of the compound carbon dots group was not significantly different (ns) compared with the blank control group (0 mg / ml). This result demonstrates that the traditional Chinese medicine compound carbon dots of this invention have no significant cytotoxicity at conventional effective concentrations, good biocompatibility, and meet the safety requirements for in vivo experiments. In the coordinate system, the vertical axis represents cell survival rate in percentage (%), and the horizontal axis represents concentration in mg / ml.

[0062] Experimental Example 3: In vivo efficacy evaluation of traditional Chinese medicine compound carbon dot therapy for granulomatous mastitis This study evaluated the in vivo therapeutic effect of the prepared traditional Chinese medicine compound carbon dots on granulomatous lobular mastitis (GLM) in rats.

[0063] 1. Animal experiments (1) Animal modeling: Referring to the method for establishing a classic rat model of granulomatous lobular mastitis, human granulomatous mastitis specimens were randomly selected, preserved in liquid nitrogen, and transported to the laboratory. The sealed tissue specimens were then thawed in a 37°C constant temperature water bath. After thawing, the tissues in the frozen tubes were removed, weighed, and manually ground until there were no obvious large particles. They were then mixed with physiological saline at a ratio of 1:3, placed in centrifuge tubes, and placed on ice. Cell homogenization was performed using a handheld ultrasonic cell lysis device to release the active components in the tissue specimen cells into the homogenate. The homogenate after cell homogenization was centrifuged at 12000 r / min for 15 min, with a centrifugation radius of 6.5 cm. The supernatant was placed in a sterile specimen bottle and mixed with complete Freund's adjuvant at a volume ratio of 1:1 to prepare an oil-in-emulsion suspension (the mixing process was carried out on ice for about 20 min, maintaining the ambient temperature between 0 and 4°C to preserve the activity of complete Freund's adjuvant). The prepared suspension was then set aside. Rats were anesthetized by intraperitoneal injection of 3% pentobarbital solution (30 mg / kg). After anesthesia, the rats were fixed to a wooden board, and the hair on the third and fourth pairs of mammary glands was removed. The skin was disinfected with 75% alcohol. The nipples were lifted with forceps, and 0.2 ml each of the prepared oil-emulsion suspension and physiological saline were injected into the third and fourth pairs of mammary glands of the rats using an inoculation syringe. This completed the artificial implantation and embedding process.

[0064] (2) Fifteen GLM rats that had successfully developed the model were randomly selected and divided into a model control group (referred to as the model group), a compound carbon dot group (also referred to as the compound CDs group), and a positive control group (triamcinolone acetonide), with five rats in each group; another five healthy rats from the same batch that had not yet developed the model were selected as the blank control group (referred to as the blank group), for a total of 20 rats. The blank control group and the model control group were injected locally with 0.2 mL of physiological saline in each breast once a day; the compound carbon dot group was injected with 0.2 mL of compound carbon dot aqueous solution per side (20 mg / kg) once a day; the positive control group was injected with 0.2 mL of triamcinolone acetonide injection per side (0.67 mg / kg) once a week. All the above groups were intervened for 14 consecutive days.

[0065] It should be noted that all animal experiments involved in the embodiments of the present invention have been approved by the medical ethics committee of our unit and have been conducted in accordance with relevant ethical standards, with ethics number HN-LL-YJSLW-2026-94.

[0066] 2. Experimental Observation (1) Staining observation of pathological morphological changes: The third and fourth pairs of mammary gland tissues of rats were dehydrated, embedded, and serially sectioned. Each section was 4 μm thick. After drying, the tissues were dewaxed, stained with hematoxylin, dehydrated again, cleared with xylene, and mounted. The pathological changes of mammary gland tissues in each group were observed under an optical microscope.

[0067] Please see Figure 12 As shown, Figure 12 The pathological H&E staining images of rat mammary tissue from each group are shown, where A is the blank group, B is the model group, C is the compound carbon dot group, and D is the positive control group. Figure 12 The lower left corner of A, B, C, and D in the diagram is marked with scales of 0, 40, 80, 120, 160, and 200 μm.

[0068] Depend on Figure 12 As observed, normal rat mammary gland tissue shows that the mammary lobules are composed of terminal ducts, acini, and interlobular stroma, surrounded by fibrous connective tissue, with a clear and orderly structure. Compared with the normal group, the mammary gland tissue of GLM rats in the model group exhibits disordered structure. Microscopically, granuloma formation centered on the lobules is visible, accompanied by infiltration of epithelioid cells, multinucleated giant cells, lymphocytes, plasma cells, neutrophils, etc., and lipid vacuoles are also observed. Compared with the model group, no obvious granuloma pathological structure was observed in the compound carbon dot group and the positive control group, and the infiltration of inflammatory cells such as lymphocytes and plasma cells was significantly reduced, as was the epithelial cell proliferation.

[0069] (2) ELISA detection of related inflammatory factor expression: ELISA was used to detect the serum immune status and related inflammatory factor expression in each group. Blood was collected from the abdominal aorta of rats, centrifuged, and the supernatant was stored at -20℃ for later use. The expression levels of inflammatory factors such as PRL, IL-6, IL-17, TNF-α, and TGF-β in rat serum were detected using an ELISA kit.

[0070] Please see Figure 13 As shown, Figure 13 The ELISA results of relevant inflammatory factors in the serum of rats in each group are shown. Figure 13 It can be known that: Prolactin (PRL): Compared with the control group, the serum prolactin (PRL) level in the model group rats was significantly increased (P<0.01), indicating successful establishment of the hyperprolactinemia model. Compared with the model group, the PRL level in the positive control group was significantly decreased (P<0.05), and the difference was statistically significant. Although the compound CDs group showed a decreasing trend compared with the model group, the difference was not statistically significant (P>0.05).

[0071] Interleukin-6 (IL-6): Compared with the control group, the IL-6 level in the model group was significantly increased (P<0.0001). After intervention with compound CDs and positive control drugs, the IL-6 levels in both the compound CDs group and the positive control group were significantly lower than those in the model group (both P<0.0001). There was no statistically significant difference between the compound CDs group and the positive control group (P>0.05), suggesting that compound CDs has a downregulating effect on IL-6 levels comparable to that of positive control drugs.

[0072] Interleukin-17 (IL-17): Compared with the blank group, the IL-17 level in the model group was significantly increased (P<0.001). After intervention with compound CDs and positive control drugs, the IL-17 level in the compound CDs group was significantly lower than that in the model group (P<0.05), and the IL-17 level in the positive control group was lower than that in the model group, but the difference was not statistically significant (P>0.05).

[0073] Transforming growth factor-β (TGF-β): Compared with the blank group, TGF-β in the model group was significantly decreased (P<0.0001). After intervention, TGF-β in the positive control group was significantly increased compared with the model group (P<0.01), and TGF-β in the compound CDs group was also increased compared with the model group, but the difference was not statistically significant (P>0.05).

[0074] Tumor necrosis factor-α (TNF-α): After modeling, TNF-α in the model group was significantly higher than that in the control group (P<0.0001). After treatment, TNF-α in both the compound CDs group and the positive control group was significantly lower than that in the model group (P < 0.0001), suggesting that compound CDs can effectively reverse the abnormal changes of this inflammatory marker.

[0075] In summary, the present invention has the following advantages: (1) Traditional Chinese medicine empowers and enhances efficacy: The carbon dot technology for compound traditional Chinese medicine (TCM) formulas not only enhances the pharmacological activity of the original formulas, thus overcoming the challenge of poor oral absorption of many TCM components, but also, through their nanoscale size (typically less than 10 nm), allows TCM carbon quantum dots to more effectively cross biological barriers, achieving precise targeted delivery to lesions by binding with drug molecules. TCM external application formulas, previously limited to oral administration, can now be formulated for local carbon dot injection, significantly improving the convenience and effectiveness of local drug delivery and expanding its application scenarios.

[0076] (2) Safety and stability: Multifaceted biocompatibility: Carbon dots in traditional Chinese medicine compound preparations typically inherit the characteristics of natural carbon sources and have undergone cytotoxicity testing. While retaining or even enhancing efficacy, they significantly reduce potential systemic toxicity and side effects, making the drugs safer. They exhibit low toxicity, high water solubility, and excellent biocompatibility, laying a clinical foundation for their treatment of granulomatous mastitis.

[0077] (3) Green, economical and sustainable: The preparation of traditional Chinese medicine compound prescriptions often results in the waste of a large amount of medicinal residue. Using this residue as a carbon source to prepare carbon dots not only turns waste into treasure and achieves high-value utilization of medicinal resources, but also provides a new approach for their green and sustainable development. Compared with common preparation methods (such as hydrothermal methods) and complex processes, this method is usually low-cost, simple to operate, and has significant potential for large-scale industrial production.

[0078] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application 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. Such 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 this application, and should all be included within the protection scope of this application.

Claims

1. A traditional Chinese medicine compound carbon dot formula for treating granulomatous mastitis, characterized in that, The raw materials for preparing the carbon dots of the traditional Chinese medicine compound include, by weight: Astragalus membranaceus 30 parts, Curcuma zedoaria (processed with vinegar) 15 parts, Smilax glabra 30 parts, Alisma plantago-aquatica 10 parts, Lonicera japonica 10 parts, Forsythia suspensa 10 parts.

2. A method for preparing carbon dots of a traditional Chinese medicine compound for treating granulomatous mastitis as described in claim 1, characterized in that, Includes the following steps: Step 1, Mixing and Calcination: The Astragalus membranaceus, Curcuma zedoaria, Smilax glabra, Alisma plantago-aquatica, Lonicera japonica and Forsythia suspensa weighed according to the above weight proportions are mixed and placed in a crucible and sealed. Then, they are calcined in a muffle furnace at 350°C. After cooling, the medicine in the crucible is ground into powder. Step 2, Boiling in a water bath: Mix the obtained powder with deionized water and boil it in a water bath; Step 3, Filtration and Dialysis: The extract is filtered to remove residues, the solution is concentrated and then dialyzed to obtain the carbon dots of the Chinese herbal compound.

3. The method for preparing carbon dots of traditional Chinese medicine compound according to claim 2, characterized in that, In step one, the crucible is covered with aluminum foil to form a seal.

4. The method for preparing carbon dots of traditional Chinese medicine compound according to claim 2, characterized in that, In step two, the powder is mixed with 3 liters of deionized water and boiled twice in a water bath, each time for 1 hour.

5. The method for preparing carbon dots of traditional Chinese medicine compound according to claim 2, characterized in that, In step three, a filter membrane with a pore size of 0.22 μm is used for filtration.

6. The method for preparing carbon dots of traditional Chinese medicine compound according to claim 2, characterized in that, In step three, dialysis is performed using a dialysis bag with a molecular weight cutoff of 1 kDa in deionized water for 168 hours.

7. The application of the traditional Chinese medicine compound carbon dots as described in claim 1 in the preparation of a drug for treating granulomatous mastitis.

8. The application according to claim 7, characterized in that, The drug is an aqueous solution and is used for local injection into granulomatous mastitis lesions.

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