Application of berberine-loaded herbal carbon dots in ulcerative colitis

Abstract

This invention discloses the application of berberine-loaded herbal carbon dots in the preparation of drugs for treating ulcerative colitis. The BLCDs of this invention exert their therapeutic effect through a dual synergistic mechanism: directly scavenging reactive oxygen species through surface oxygen-containing functional groups, and simultaneously inhibiting the NLRP3 inflammatory pathway and downstream pro-inflammatory factors by promoting IL-10 expression. In a DSS-induced ulcerative colitis mouse model, BLCDs showed superior efficacy compared to the positive control drug 5-ASA and its single component, significantly improving intestinal inflammation, oxidative stress, and barrier dysfunction. Their therapeutic advantage stems from the intestinal retention effect conferred by their larger particle size, as well as their regulatory effects on intestinal flora and metabolites, particularly by inhibiting Klebsiella to reduce TMAO production, thereby activating the multi-cascade regulatory network of IL-10.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine application technology, specifically relating to the application of herbal carbon dots loaded with berberine in ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a chronic inflammatory bowel disease of unknown etiology, primarily affecting the colorectal mucosa and submucosa. Characterized by recurrent flare-ups and a protracted course, it is often referred to as "green cancer." The pathogenesis of UC is complex, mediated by a combination of genetic factors, immune disorders, gut microbiota dysbiosis, and environmental factors. Currently, there is no cure, and the disease carries a risk of malignant transformation. In recent years, influenced by lifestyle changes and improved diagnostic capabilities, the prevalence of UC has continued to rise, becoming a significant public health issue.

[0003] Berberine (BBR) is the main active ingredient in Coptis chinensis, and its molecular formula is C6H2O. 20 H 18 NO4 + Berberine carries a positive charge due to the presence of a quaternary ammonium nitrogen atom in its structure. The multi-target pharmacological effects of berberine in the treatment of ulcerative colitis have attracted considerable attention. Studies have shown that berberine (BBR) alleviates colitis through multiple mechanisms, primarily including regulating the expression of pro-inflammatory cytokines, enhancing intestinal epithelial barrier function by upregulating tight junction proteins, modulating gut microbiota composition, and intervening in key signaling pathways such as NF-κB, Nrf2, EIF2AK2, JAK / STAT, AKT, and NLRP3. Furthermore, BBR can reduce oxidative stress damage in colonic tissue and inhibit the release of inflammatory mediators. Despite exhibiting these favorable pharmacological activities, the clinical application of BBR still faces challenges such as poor water solubility, low oral bioavailability, and insufficient accumulation in the inflamed colon. Therefore, developing novel formulation strategies to overcome these limitations has become an important direction for promoting the clinical translation of BBR.

[0004] Traditional Chinese medicine (TCM) is a rich treasure trove of pharmacological resources. Carbonized herbs, as unique products of this process, have been used clinically for over a thousand years due to their traditional effects such as anti-inflammation, hemostasis, and antidiarrheal properties. Recent studies have discovered that TCM carbon dots (TCM-CDs) are key active nanocomponents in carbonized herbs. Prepared through high-temperature heating of herbs, they exhibit enzyme-like effects, displaying activities similar to natural oxidases (OXD), glucose oxidase (GOD), peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD). Notably, in addition to their efficient scavenging of reactive oxygen species in vivo, TCM-CDs also demonstrate good biocompatibility and in vivo safety, showing significant potential value in antioxidant and anti-inflammatory clinical applications.

[0005] The self-assembly of active molecules in traditional Chinese medicine (TCM) formulas is a cutting-edge research area in the modernization of TCM in recent years. This process refers to the spontaneous formation of ordered, structurally stable, and functionally specific nanoscale aggregates by certain active components in TCM (such as flavonoids, alkaloids, and saponins) through weak interactions such as non-covalent bonds during decoction or after entering the body. Currently, most reports focus on the self-assembly between active small molecules; the interaction and synergistic mechanisms between nanomaterials such as TCM-CDs and active small molecules remain to be elucidated. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide the application of herbal carbon dots loaded with berberine in the preparation of a medicament for treating ulcerative colitis, wherein the herbal carbon dots are carbon dots from Rehmannia glutinosa.

[0007] Technical solution: In order to solve the above-mentioned technical problems, the present invention provides the application of herbal carbon dots loaded with berberine in the preparation of drugs for treating ulcerative colitis.

[0008] The herbal carbon dots loaded with berberine are obtained by self-assembling Rehmannia glutinosa carbon dots with berberine.

[0009] The mass ratio of the carbon dots of *Rehmannia glutinosa* to berberine is 1:5 to 5:1.

[0010] The carbon dots of *Ulmus pumila* were prepared using a one-step hydrothermal method.

[0011] The one-step hydrothermal method specifically includes the following steps: adding raw Rehmannia glutinosa powder and pure water in a mass-volume ratio of 1:1 to 1:10 g / mL into a reaction vessel and reacting at 150 to 300°C for 4 to 12 hours; after the reaction, filtering, dialysis, and vacuum freeze-drying are performed to obtain raw Rehmannia glutinosa carbon dots.

[0012] Preferably, the mass-to-volume ratio of the raw Sanguisorba officinalis powder to pure water is 1:10 g / mL, the hydrothermal reaction temperature is 200℃, and the reaction time is 8 hours.

[0013] The average particle size of the carbon dots in the *Ulmus pumila* species is 8.5 ± 0.35 nm.

[0014] The carbon point concentration of the raw *Ulmus pumila* is 1~100 µg / mL.

[0015] The dosage form of the drug includes liquid preparations, tablets, capsules, or dry suspensions.

[0016] The drug is one that upregulates IL10 gene expression and / or inhibits the expression of the NLRP3 inflammasome pathway and its downstream pro-inflammatory factors.

[0017] The drug in question is used to improve intestinal inflammation, oxidative stress, and barrier dysfunction.

[0018] The drug is one that enhances the abundance and diversity of intestinal flora, and / or inhibits the growth of harmful bacteria such as Klebsiella spp., and / or reduces the formation of trimethylamine oxide.

[0019] The dosage of the raw Rehmannia glutinosa carbon dots is 1~100 mg / kg mice.

[0020] In this invention, carbon dots (LCDs) derived from *Sanguisorba officinalis* were synthesized via a one-step hydrothermal method and then self-assembled with berberine through electrostatic interactions and hydrogen bonding to construct novel nanomedicines, BLCDs. This invention systematically investigated the SOD-like activity and reactive oxygen species scavenging mechanism of LCDs, and explored the synergistic anti-inflammatory effect of BBR and LCDs after self-assembly. Through RNA-seq, 16S rDNA, non-target metabolomics analysis, and cell, animal, and intestinal organoid (CNO) models, the mechanism by which BLCDs improve ulcerative colitis (UC) by regulating IL10 and TMAO was systematically elucidated.

[0021] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: This invention successfully prepares uniformly sized carbon dots (LCDs) from *Sanguisorba officinalis* using a one-step hydrothermal method. The LCDs are rich in oxygen-containing functional groups such as carboxyl, hydroxyl, and carbonyl groups, exhibiting superoxide dismutase (SOD)-like activity. After self-assembling LCDs with berberine (BBR) to construct nanocomplexes (BLCDs), they still possess SOD-like activity and show significantly superior anti-inflammatory and antioxidant effects compared to single-component formulations in a colitis cell model. Transcriptomic analysis shows that BLCDs treatment significantly upregulates IL10 gene expression, thereby inhibiting the expression of the NLRP3 inflammasome pathway and its downstream pro-inflammatory factors. In a DSS-induced ulcerative colitis mouse model, BLCDs, due to their larger particle size and intestinal retention effect, effectively improved intestinal inflammation, oxidative stress, and barrier dysfunction. 16S rDNA sequencing results showed that BLCDs could enhance the abundance and diversity of gut microbiota, inhibit the growth of harmful Klebsiella spp., and reduce the production of trimethylamine oxide (TMAO). Further intestinal organoid experiments confirmed that BLCDs reversed TMAO-induced inflammatory lesions through IL10. In vitro and in vivo safety assessments demonstrated that BLCDs possessed good biocompatibility. In summary, this invention successfully constructed a BLCDs nanocomposite possessing both the antioxidant activity of LCDs and the anti-inflammatory function of BBR, which improves ulcerative colitis by regulating IL10 expression and the gut microbiota-TMAO metabolic pathway. This discovery provides a new scientific perspective for understanding the compatibility mechanisms of traditional Chinese medicine compound formulations and lays the foundation for the development of nanomedicines based on the active components of traditional Chinese medicine. Attached Figure Description

[0022] Figure 1 Synthesis and characterization of BLCDs. (A) Schematic diagram of BLCD synthesis. (B) TEM image of LCDs, with inset showing particle size distribution. (C) TEM image of BLCDs, with inset showing particle size distribution. (D) Hydrated particle size distribution of LCDs, BBR, and BLCDs. (E) Zeta potential diagrams of LCDs, BBR, and BLCDs. (F) FT-IR diagrams of LCDs, BBR, and BLCDs. (G) UV-Vis absorption spectra of LCDs, BBR, and BLCDs. (H) Photographs of LCDs, BBR, and BLCDs powders and their aqueous solutions under sunlight and UV light. (I) Excitation and emission spectra of LCDs, BBR, and BLCDs. (J) XPS full spectrum and high-resolution C1s, N1s, and O1s spectra of LCDs. (K) shows the XPS full spectrum and high-resolution C1s, N1s, and O1s spectra of BLCDs.

[0023] Figure 2 The figures show the electron diffraction pattern, X-ray diffraction pattern, Raman spectroscopy analysis, and loading rate of the complex formed by BBR and LCDs for LCDs; (A) is the selected area electron diffraction (SEAD) pattern of LCDs, with the TEM measurement area in the lower right corner inset. (B) is the X-ray diffraction pattern of LCDs. (C) is the Raman spectroscopy analysis data of LCDs. (D) is the loading rate of BBR on the complex formed by BBR and LCDs with different mass ratios.

[0024] Figure 3 The following graphs show the ROS scavenging results of BLCDs: (A) Schematic diagram of LCD modification; (B) Changes in SOD-like activity of LCDs before and after passivation, reduction, and re-oxidation treatments; (C) Results of total ROS scavenging capacity of LCDs and BLCDs assessed by the ABTS method; (D) Results of •OH scavenging activity determined by the salicylic acid method; (E) Results of •O2 scavenging activity assessed by the pyrogallol autoxidation method. -The results of the clearance efficiency are shown in the figure. (F) is a schematic diagram of the anti-ROS mechanism of BLCDs. (G) is a DCFH fluorescence staining image of NCM460 cells stimulated with LPS after treatment with LCDs, BBR, and BLCDs (50 μg / mL). The PBS group only added PBS. (H) is a graph showing the quantitative analysis results of ROS levels in NCM460 cells stimulated with LPS after treatment with LCDs, BBR, and BLCDs (50 μg / mL). (I) RT-qPCR results of mRNA levels of inflammatory factors IL6, TNFα, IL1α, and IL1β in LPS-stimulated NCM460 cells treated with LCDs, BBR, and BLCDs (50 μg / mL); (J) ELISA results of protein levels of inflammatory factors IL6, TNFα, IL1α, and IL1β in LPS-stimulated NCM460 cells treated with LCDs, BBR, and BLCDs (50 μg / mL); (K) AO & EB staining images of LPS-stimulated NCM460 cells treated with LCDs, BBR, and BLCDs (50 μg / mL); (L) Statistical data of cell viability of LPS-stimulated NCM460 cells treated with LCDs, BBR, and BLCDs (50 μg / mL) as detected by CCK8 assay.

[0025] Figure 4 Cell viability graphs after treatment with different concentrations of LCDs and BLCDs; (A) Cell viability graphs of different concentrations of LCDs. (B) Cell viability graphs of different concentrations of BLCDs.

[0026] Figure 5 RNA-seq analysis of LPS-induced NCM460 cells before and after BLCDs treatment. (A) Volcano plot of differentially expressed genes (DEGs). LPS: LPS-induced NCM460 cells; BLCDs: BLCDs-treated NCM460 cells; BLCDs.LPS: LPS-induced NCM460 cells treated with BLCDs. (B) Venn diagram analysis results. (C) GO and KEGG enrichment analysis results of upregulated differentially expressed genes. (D) GO and KEGG enrichment analysis results of downregulated differentially expressed genes.

[0027] Figure 6Figure 1 shows the changes in different factors in NCM460 cells before and after BLCDs treatment; (A) Radar graph showing changes in the transcriptome of IL10-related pathways in NCM460 cells before and after BLCDs treatment; (B) RT-qPCR results showing the mRNA and protein expression levels of IL10 in NCM460 cells after treatment with different reagents; (C) RT-qPCR results showing the mRNA and protein expression levels of NLRP3 in NCM460 cells after treatment with different reagents; (D) ELISA results showing the expression levels of IL1β and IL18 in NCM460 cells after treatment with different reagents; (E) ELISA results showing the expression levels of TNFα and IL6 in NCM460 cells after treatment with different reagents: The PBS group involved treatment with only PBS buffer in the cells; the LPS group involved induction with only LPS; the BLCDs group involved NCM460 cells without LPS, only with BLCDs; and the BLCDs+LPS group involved induction with LPS followed by the addition of BLCDs.

[0028] Figure 7 The following graphs show the changes in RAW264.7 cells under different treatment conditions: (A) ELISA results of IL10 expression level; (B) ELISA results of NLRP3 expression level; (C) ELISA results of IL1β and IL18 expression levels; (D) ELISA results of TNFα and IL6 expression levels; (E) ROS fluorescence intensity results; (F) Cell viability results. JES5-2A5 is an anti-mouse IL10 IgG1 monoclonal antibody used at a final concentration of 5 μg / mL, co-incubated with BLCDs (50 μg / mL) for 48 hours.

[0029] Figure 8 The figure shows the effect of different concentrations of BLCDs on the DSS-induced disease activity index score in mice.

[0030] Figure 9 Figures show the therapeutic effects of BLCDs on DSS-induced mice. (A) Schematic diagram of BLCDs treatment in animals. (B) Visual diagram of the dissected colon. (C) Diagram of the anus of mice before and after treatment. (D) Diagram of mouse weight change. (E) Diagram of disease activity index score results. (F) Diagram of colon length statistical analysis results. (G) H&E staining of colon in each group of mice. Black boxes indicate magnified areas. (H) AB staining of colon in each group of mice. Black boxes indicate magnified areas. (I) PSR staining of colon in each group of mice. Black boxes indicate magnified areas. (J) Diagram of pathological scoring results. (K) Diagram of quantitative analysis results of goblet cells in colonic crypts in each group. (L) Diagram of quantitative results of colonic fibrosis area in mice.

[0031] Figure 10 Figure 1 shows the therapeutic mechanism analysis of BLCDs. (A) Immunofluorescence staining of DAPI, ZO-1, and Occludin in colon tissues of each group. (B) Quantitative analysis of ZO-1 and Occludin expression levels. (C) DHE / DAPI staining of colon tissues of each group. (D) Quantitative analysis of the DHE / DAPI fluorescence intensity ratio in colon tissues of each group. (E) Immunohistochemical staining of IL10 and NLRP3 in colon tissues of each group. (F) Expression levels of IL10 and NLRP3. (G) ELISA detection results of IL1β and IL18 expression levels. (H) ELISA detection results of IL6 and TNFα expression levels. (I) Near-infrared fluorescence imaging of major mouse tissues of LCDs and BLCDs at different time points (0, 1, 3, 6, 12, 24 h). (J) Biodistribution of LCDs and BLCDs at 24, 48, and 72 h.

[0032] Figure 11 H&E staining images of major tissues in mice from different treatment groups.

[0033] Figure 12 The following figures illustrate the effects of BLCDs on the gut microbiota induced by DSS in mice. (A) The number of OTUs observed in mice under different treatment groups; (B) The Shannon index results for mice under different treatment groups; (C) The abundance of mice under different treatment groups; (D) The gut microbiota community assessed by NMDS2 analysis in mice under different treatment groups; (E) The relative abundance (percentage of total sequences) at the genus level in mice under different treatment groups; (F) The LDA scores for the BLCDs group and the DSS group; (G) The relative abundance of *Lactobacillus*, *Lactobacillus*, *Ackermania*, *Escherichia coli*-*Shigella*, and *Klebsiella* in different treatment groups.

[0034] Figure 13Figures showing the effects of BLCDs on DSS-induced intestinal metabolites in mice: (A) OPLS-DA plot; (B) Heat map of differential metabolite aggregation between the DSS and BLCDs groups; (C) ELISA results of trimethylamine levels in feces and plasma; (D) ELISA results of trimethylamine oxide levels in feces and plasma; (E) Bright-field images and H&E staining of CNOs treated with PBS, BLCDs, LPS, TMAO, LPS+BLCDs, and TMAO+BLCDs; (F) Immunohistochemical staining results of IL10 and NLRP3 in each group of CNOs; (G) ELISA results of IL10 expression level; (H) ELISA results of NLRP3 expression level; (I) ELISA results of IL1β and IL18 expression levels; (J) ELISA results of TNFα and IL6 expression levels.

[0035] Figure 14 Figure 1 shows the diameter, viability, and MDA level of normal colonic organoids (CNOs); (A) shows the diameter, (B) shows the viability, and (C) shows the malondialdehyde (MDA) level in the culture medium.

[0036] Figure 15 The images show the immunohistochemical results and expression levels of IL10 in CNOs under various treatments, as well as other results. (A) Immunohistochemical results of IL10 in CNOs under various treatments; (B) ELISA results of IL10 expression levels; (C) Bright-field image of CNOs; (D) Diameter result; (E) Viability result; (F) MDA level result in the culture medium.

[0037] Figure 16 The following figures represent the results of animal experiments: (A) Schematic diagram of animal safety experiments; (B) Changes in mouse body weight; (C) H&E staining of major tissues; (D) Complete blood count results of mice; (E) Liver and kidney function indicators of mice; (F) Hemolysis rate results of BLCDs. Detailed Implementation

[0038] Example 1: Synthesis of LCDs and BLCDs

[0039] Take 5 g of raw Rehmannia glutinosa (purchased from Anhui Huchuntang Chinese Herbal Pieces Co., Ltd., batch number: 250506, origin: Jiangsu), and pulverize it into powder using a grinder. Then, add the Rehmannia glutinosa powder and ddH2O to a reaction vessel at a ratio of 1:10 (w / v, g / mL), and heat at 200°C for 8 hours. After the reaction is complete, filter the product, dialyze it using a 1000 D dialysis bag, and freeze-dry it under vacuum at -50°C. The resulting product is named LCDs and is stored long-term at 4°C.

[0040] The prepared LCDs were mixed with berberine (BBR, MCE, HY-N0716) in different mass ratios (1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1). When the two were mixed at a mass ratio of 1:2, the loading rate of the LCDs on the BBR was the highest.

[0041] Subsequent experiments were conducted at a mass ratio of 1:2. The mixed powders were then dissolved in ddH₂O at a ratio of 1:10 (w / v, g / mL) and transferred to a reaction vessel for hydrothermal reaction. After heating at 100°C for 1 hour, the reaction product was filtered, dialyzed using a 1000 D dialysis bag, and then freeze-dried under vacuum at -50°C. The resulting final product was named BLCDs and stored long-term at 4°C.

[0042] Example 2: Characterization and Application of LCDs and BLCDs

[0043] 1. Nanoparticle size, morphology observation and particle size / potential analysis, elemental and chemical state analysis and structural analysis

[0044] Morphological observation and particle size / potential analysis of LCDs and BLCDs were performed using transmission electron microscopy (TEM, FEI Tecnai F20) and a nanoparticle size and zeta potential analyzer (Malvern Zetasizer Nano ZS90). Spectroscopic characterization was conducted using Fourier transform infrared spectroscopy (FT-IR, Nexus 670), ultraviolet-visible spectrophotometry (UV-Vis, PE Lambda 950), and fluorescence spectroscopy (FLS 980). Elemental and chemical state analysis was performed using X-ray photoelectron spectroscopy (XPS, Thermo, Kalpha). Furthermore, the structure of LCDs was further elucidated using Raman spectroscopy (Raman, Thermo, DXR) and X-ray diffraction (XRD, D8).

[0045] 2. Reactive oxygen species detection

[0046] In the ABTS experiment, 5 mL of 2,2'-hydrazine bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) solution (concentration 7.4 mmol / L, Sigma, A9941) was mixed with 88 μL of K2S2O8 solution (concentration 2.6 mmol / L) and allowed to stand at room temperature in the dark for 16 h to obtain the ABTS working solution. The ABTS working solution was diluted 30 times with PBS (pH 7.4, 0.01 mol / L) to achieve a suitable absorbance at 734 nm. Take 0.2 mL of diluted ABTS (approximately 0.2 mmol / L) working solution, add 10 μL of LCDs or BLCDs solution (both with a concentration of 50 μg / mL), mix well, and place in a microplate reader (BioTek, Winooski, VT, USA). Measure the absorbance at 734 nm at time points of 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h to evaluate the sample's scavenging activity against ABTS free radicals.

[0047] The salicylic acid (SA) method is based on the Fenton reaction detection system, in which hydroxyl radicals (•OH) react with SA to generate 2,3-dihydroxybenzoic acid (2,3-DHBA), which exhibits characteristic UV absorption at 510 nm. When LCDs or BLCDs with •OH scavenging activity are added (final concentrations of 0, 10, 25, 50, and 100 μg / mL, respectively), the number of •OH radicals in the system decreases, resulting in a corresponding decrease in absorbance at 510 nm. The reaction mixture (containing SA, FeSO4, LCDs / BLCDs, and H2O2) was diluted with PBS and incubated at 37°C for 20 min, followed by measurement of the absorbance at 510 nm using a UV-Vis spectrophotometer.

[0048] In the pyrogallol method, 10 μL of LCDs or BLCDs solution (final concentration of 50 μg / mL) was added to 2940 μL of Tris-HCl buffer (0.05 mol / L, pH 7.4) containing 1 mmol / L Na2EDTA. Then, 50 μL of pyrogallol solution (prepared with 1 mmol / L HCl, concentration 60 mmol / L) was added, and the mixture was immediately and rapidly shaken at room temperature. The absorbance of the mixture at 325 nm was measured using a spectrophotometer (FLS 980).

[0049] In the DCFH staining experiment, 1×10 6NCM460 cells (purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were seeded in 6-well plates and cultured for 24 h. Subsequently, lipopolysaccharide (LPS) (20 μg / mL, MCE, HY-D1056) was added to the cells for 24 h. After induction, LCDs (final concentration 50 μg / mL), BBR (final concentration 50 μg / mL), or BLCDs (final concentration 50 μg / mL) were added to the cells for co-incubation for 24 h. Finally, 10 μmol / L DCFH-DA working solution (MCE, HY-D0940) was added to the cells, and the cells were incubated at 37°C and 5% CO2 in the dark for 30 min. After washing away excess dye with PBS, the intracellular reactive oxygen species (ROS) levels were observed under a fluorescence microscope and quantitatively analyzed using a BioTek microplate reader.

[0050] 3. Cell viability detection

[0051] The cell lines used in this invention were purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, including NCM460 (normal human intestinal epithelial cells) and RAW264.7 (mouse mononuclear macrophage leukemia cells). RAW264.7 cells were cultured in DMEM medium, and NCM460 cells were cultured in 1640 medium. All media were supplemented with 10% fetal bovine serum (HyClone), 100 U / mL penicillin (Thermo Fisher), and 100 μg / mL streptomycin (Thermo Fisher), and cultured in a humidified incubator at 37°C and 5% CO2. Cell viability was assessed using the CCK-8 assay kit (Yeasen). NCM460 cells were cultured at 5 × 10⁻⁶ cells / mL. 3 Seeds were planted at a density of 100 cells / well in 96-well plates and incubated overnight. Then, LPS (final concentration 20 μg / mL) was added for induction treatment for 24 h. After induction, LCDs (final concentration 50 μg / mL), BBR (final concentration 50 μg / mL), or BLCDs (final concentration 50 μg / mL) were added to the wells. After incubation, 10 μL of CCK-8 reagent was added to each well, and incubation continued for 1 h. The absorbance at 450 nm was measured using a BioTek microplate reader.

[0052] 4. Acridine orange-ethidium bromide (AO&EB) staining

[0053] Cells in the logarithmic growth phase were taken and analyzed at a concentration of 1 × 10⁻⁶. 5Cells were seeded at a density of cells / well in 24-well plates and cultured overnight. Then, LPS (final concentration 20 μg / mL) was added for 24 h induction. After treatment, LCDs (final concentration 50 μg / mL), BBR (final concentration 50 μg / mL), or BLCDs (final concentration 50 μg / mL) were added to the test cells. After 48 h of incubation, all cells were stained with acridine orange & ethidium bromide (AO & EB) staining solution (Sangon, E607308). Following the kit instructions: the culture medium was aspirated, PBS buffer was added, and then 10 µL of AO (final concentration 1 μg / mL) and 10 µL of EB (final concentration 1 μg / mL) were added per 180 µL of PBS buffer (pH 7.4, 0.01 mol / L) for staining. The cells were incubated at room temperature in the dark for 5 min. Live cells appeared uniformly green, while necrotic cells were stained orange. Cells were imaged under a fluorescence microscope to observe live and dead cells.

[0054] 5. RNA sequencing analysis

[0055] RNA sequencing (RNA-Seq) was performed by Megagene Corporation, with three biological replicates for each treatment group. A total of 12 RNA samples were sequenced and analyzed, including RNA samples extracted independently from four groups of NCM460 cells: PBS group, BLCDs group, and LPS-induced groups before and after BLCDs treatment. After obtaining transcript readings, the transcript readings were converted into gene readings using the R package tximport. Subsequently, differential gene expression analysis was performed using the DESeq2 software. Differentially expressed genes (DEGs) were selected based on FDR < 0.05 and |fold change| ≥ 1.5 as screening criteria, and then GeneOntology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted.

[0056] 6. Enzyme-linked immunosorbent assay (ELISA)

[0057] The following kits were used for ELISA assays: IL6 ELISA kit (Abcam, human ab178013, mouse ab222503), TNFα ELISA kit (Abcam, human ab181421, mouse ab208349), IL1β ELISA kit (Abcam, human ab214025, mouse ab197742), IL1α ELISA kit (Abcam, human ab100560), IL10 ELISA kit (Abcam, human ab185986, mouse ab255729), NLRP3 ELISA kit (Abcam, human ab274401, mouse ab279417), and IL18 ELISA kit (Abcam, human ab215539, mouse ab216165). The main steps included preparing the reagents according to the instructions and allowing them to reach room temperature. Add 50 µL of the test sample to each well (the test sample is: 1×10⁻⁶ ppm of the test sample). 6 NCM460 cells were seeded per well in 6-well plates and cultured for 24 h. Subsequently, LPS (final concentration 20 μg / mL) was added to the cells for 24 h. After induction, LCDs (final concentration 50 μg / mL), BBR (final concentration 50 μg / mL), or BLCDs (final concentration 50 μg / mL) were added to the cells for co-incubation for 24 h, or different concentrations of control standards (final concentrations: 0, 15.6, 31.2, 62.5, 125, 250, 500, 1000 pg / mL) were added, followed by 50 µL of the antibody mixture from the kit (final concentration: 5 µg / mL). After incubation at room temperature for 1 h, the supernatant was discarded and the cells were washed three times with washing buffer. 100 µL of TMB substrate was added to each well, and the cells were incubated in the dark for 15 min. The reaction was then stopped by adding 100 µL of stop solution, and the OD value was immediately read at 450 nm.

[0058] 7. RT-qPCR

[0059] Cells were lysed using 1 mL TRIzol (Sigma) (1×10⁻⁶ cells per cell). 7RNA was extracted from the aqueous phase of the sample by adding 200 µL of chloroform. The RNA was then precipitated with 1 mL of isopropanol and washed successively with 200 µL of 75% ethanol. The RNA precipitate was dissolved in 100 µL of ddH2O, and the total RNA concentration was determined using Nanodrop. The entire process must be performed under RNase-free conditions to ensure no RNA degradation. cDNA was synthesized by reverse transcription using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, R211-01), followed by real-time quantitative PCR amplification using the HiScript II One Step RT-PCR Kit (Vazyme, P611-01). The reaction mixture consisted of: 2 µL upstream primer, 2 µL downstream primer, 25 µL One Step Mix, 2.5 µL One Step Enzyme Mix, 8.5 µL cDNA, and 10 µL ddH2O. The reaction program was as follows: 94°C for 3 min; followed by 35 cycles of 94°C for 30 s and 60°C for 30 s. Using GAPDH as an internal reference gene, 2 -ΔΔCt The relative transcription level of mRNA was calculated using a specific method. Primer information is detailed in Table 1. All experiments were performed in triplicate to ensure the reliability of the results.

[0060] Table 1 qPCR primer sequences

[0061]

[0062] 8. Animal experiments on ulcerative colitis

[0063] In the ulcerative colitis model experiment, 30.0 g of sodium dextran sulfate (DSS) was dissolved in 1 L of water to prepare a 3% DSS solution. Eight-week-old female BALB / c mice were randomly divided into seven groups (n = 6): PBS group (healthy control group), DSS group (model group), 5-ASA group (mesalazine, positive control group), LCDs group, BBR group, BBR+LCDs group (a mixture of BBR and LCDs), and BLCDs group. Mouse weight and excretion were observed and recorded. Modeling began after 7 days of acclimatization. From day 0 of modeling, the DSS group and all drug-treated groups had free access to the 3% DSS solution, and weight and excretion were continuously monitored for 14 days. Drug administration was performed on days 7, 9, and 11 after modeling: the 5-ASA group received 100 mg / kg 5-ASA by gavage; the LCDs group received 80 mg / kg LCDs by gavage; the BBR group received 80 mg / kg BBR by gavage; the BBR+LCDs group received a mixture of BBR and LCDs by gavage (BBR 53.33 mg / kg, LCDs 26.67 mg / kg); and the BLCDs group received 80 mg / kg BLCDs by gavage. The degree of inflammation in each group of mice was assessed using the Disease Activity Index (DAI), which was calculated based on a combination of body weight, fecal characteristics, and occult blood test results. The anus of the mice was photographed before and after treatment. After treatment, the mice were euthanized, and the colon was removed and its length measured. The colon tissue was stained with hematoxylin and eosin (H&E), alexandrite blue (AB), and Sirius red (PSR).

[0064] In addition, Cy5.5 fluorescent dye was labeled onto LCDs and BLCDs using the EDC / NHS method, and the fluorescently labeled products were obtained after dialysis and vacuum drying. A standard curve was established to correlate Cy5.5 fluorescence intensity with LCDs or BLCDs concentration. Subsequently, DSS-induced ulcerative colitis model mice were administered LCDs or BLCDs (80 mg / kg) by gavage. Animals were sacrificed at 0, 1, 3, 6, 12, and 24 h after administration, and major tissues (heart, liver, spleen, lung, kidney, and colon) were dissected for near-infrared fluorescence imaging. The biodistribution of LCDs and BLCDs at 24, 48, and 72 h after administration was also detected. The tissue samples included heart, liver, spleen, lung, kidney, colon, feces, blood, and urine. The concentration of LCDs or BLCDs in the tissues was calculated based on the standard curve of Cy5.5 fluorescence intensity versus sample concentration, and the results were expressed as percentage of injected dose per gram of tissue (%ID / g).

[0065] 9. Analysis of hematoxylin-eosin (H&E), alicin blue (AB), and Sirius red (PSR) staining

[0066] H&E staining was performed on heart, liver, spleen, lung, kidney, and colon tissues from mice in each group (PBS, DSS, 5-ASA, LCDs, BBR, BBR+LCDs, and BLCDs). The procedure was as follows: paraffin sections were dewaxed and hydrated, stained with hematoxylin for 5 min, differentiated with hydrochloric acid and ethanol, blued with ammonia, stained with eosin for 3 min, dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. Under a light microscope, the cell nuclei appeared blue-purple, and the cytoplasm and extracellular matrix appeared pink.

[0067] Colon tissues from mice in each group (PBS, DSS, 5-ASA, LCDs, BBR, BBR+LCDs, and BLCDs) were stained with Alcian Blue (AB): After dewaxing and hydration, sections were stained with Alcian Blue solution at pH 2.5 for 30 min, rinsed with running water, and nucleus-fixed red lightly stained the cell nuclei. The sections were then dehydrated, cleared, and mounted. Acidic mucopolysaccharides appeared blue, and cell nuclei appeared red.

[0068] Sirius Red (PSR) staining: After bringing the required reagents and materials to room temperature, dewax and hydrate colon tissue sections, add Sirius Red staining solution and incubate for 60 min. Rinse twice quickly with acetic acid solution, dehydrate with anhydrous ethanol, clear with xylene, and mount with synthetic resin. PSR can specifically bind to collagen fibers, making them appear red under a light microscope.

[0069] 10. Immunofluorescence experiment

[0070] Colon tissue samples from mice in each group (PBS, DSS, 5-ASA, LCDs, BBR, BBR+LCDs, and BLCDs) were fixed and incubated overnight at 4°C with ZO-1 primary antibody (Abcam, ab307799, 1:500) and Occludin primary antibody (Abcam, ab216327, 1:200), respectively. After washing three times with TBST, the samples were incubated in the dark with Alexa Fluor 647-labeled secondary antibody (Abcam, ab300101, 1:100, for detecting ZO-1) or Alexa Fluor 488-labeled secondary antibody (Abcam, ab150077, 1:500, for detecting Occludin). After incubation, the samples were washed three times with TBST (5 min each time) and then stained with DAPI (Beyotime, C1005). After incubation at room temperature for 5 min, the staining solution was discarded, and the samples were washed three times with TBST (5 min each time) and observed directly under a fluorescence microscope.

[0071] 11. Culture of normal colorectal organoids

[0072] Patient-derived normal colorectal organoids (CNOs) were resuscitated from cryopreserved organoid lines. The organoids were resuspended in Matrigel and seeded into 6-well plates. Cultured using a human colon organoid culture kit (MCE, HY-K6112), the medium was changed every 48 h. Subculture was performed every 7–10 days, using a pipette tip for mechanical dispersal or, as needed, 1–2 mL of TrypLE. TM Express (GIBCO) is used for digestion.

[0073] In the CNOs experiment, LPS (final concentration 50 µg / mL) or trimethylamine oxide (TMAO) (final concentration 200 mM) was added for 24 h to induce knots. After induction, BLCDs (final concentration 50 µg / mL) were added to the test wells and incubated for 48 h. Finally, the diameter changes of CNOs were observed under a microscope.

[0074] 12. Immunohistochemical staining experiment

[0075] Immunohistochemical staining of colon tissue or CNOs sections from mice in each group (PBS, DSS, 5-ASA, LCDs, BBR, BBR+LCDs, and BLCDs) was performed using IL-10 and NLRP3. After dewaxing and hydration, the sections were placed in citrate buffer (0.1 mol / L, pH 6.0) for antigen retrieval. Endogenous peroxidase activity was blocked with 3% H2O2, and non-specific binding sites were blocked with 5% BSA. Subsequently, IL-10 primary antibody (Abcam, ab133575 to human, ab9969 to mouse, 1:200) or NLRP3 primary antibody (Invitrogen, MA5-23919, 1:100) was added, and the sections were incubated overnight at 4°C. After washing with PBS, HRP-labeled goat anti-rabbit IgG secondary antibody (Abcam, ab288151, 1:5000) was added, and the sections were incubated at room temperature for 1 h. After washing again, DAB was used for staining, and the cell nuclei were counterstained with hematoxylin. The slides were then mounted. Positive signals were observed under a light microscope.

[0076] 13. 16S rDNA sequencing analysis

[0077] Genomic DNA was extracted from feces of mice in each group (PBS, DSS, 5-ASA, LCDs, BBR, BBR+LCDs, and BLCDs) (n=3). The concentration of genomic DNA was ensured to be >50 ng / μl, the total amount >5 μg, the OD 260 / 280 to be between 1.8 and 2.0, and no obvious RNA bands were detected by electrophoresis. Clustering was performed to generate OUTs. Based on the OUTs, species classification and abundance statistics were performed, and single-sample complexity analysis, between-sample complexity analysis, and between-group significant difference analysis were conducted.

[0078] 14. Non-target metabolomics analysis

[0079] Metabolomics analysis was commissioned to Megagene Corporation (Shenzhen). The study subjects were colon tissue samples from DSS-induced mice before and after BLCDs treatment (6 biological replicates per group, designated DSS group and BLCDs group, respectively). Non-targeted metabolomics analysis was performed using LC-MS / MS. Samples were analyzed using a Vanquish ultra-high performance liquid chromatography system tandem with a Q-Exactive high-resolution mass spectrometer (Thermo). Chromatographic separation was performed using a Hypersil Gold column (100×2.1 mm, 1.9 μm, Agilent). Raw data were processed using Compound Discoverer 3.3 (Thermo) software and matched against the mzCloud, mzVault, and MassList databases to achieve relative quantification of metabolites. Orthogonal partial least squares discriminant analysis (OPLS-DA) and differential compound screening were performed on the data.

[0080] 15. In vivo safety experiments

[0081] In the safety assessment experiment, 8-week-old female BALB / c mice were randomly divided into two groups (PBS group and BLCDs group, 10 mice in each group). PBS (100 μL) or BLCDs (80 mg / kg) were injected via tail vein, respectively, and mouse body weight was monitored daily. Mice were sacrificed on day 7, and whole blood and serum samples were collected for complete blood count and liver and kidney function biochemical indicators. Simultaneously, heart, liver, spleen, lung, and kidney tissues were collected for H&E staining. Whole blood samples were analyzed using a Hematology Analyzer (Mindray, BC-60R) to detect red blood cell (RBC), white blood cell (WBC), platelet (PLT), and hemoglobin (HGB) levels. Serum samples were prepared as follows: whole blood was incubated at room temperature for 2 h, centrifuged at 300 rpm for 15 min at 4°C, and the supernatant was immediately analyzed using an Automatic Biochemical Analyzer (BIOBASE, BK-400).

[0082] Blood compatibility assessment of BLCDs was performed using 1 mL of mouse blood. Red blood cells were separated by repeated centrifugation, and 50 μL of the red blood cell suspension was mixed with 450 μL of different concentrations of BLCDs (1, 10, 20, 50, 100, 500 μg / mL). The mixture was incubated at 37°C for 3 h. Red blood cells treated with PBS and ddH2O served as negative and positive controls, respectively. After incubation, the mixture was centrifuged, and the supernatant was measured at 570 nm to determine the absorbance and calculate the hemolysis rate.

[0083] 16. Data Statistical Analysis Methods

[0084] All experimental data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis and graphing were performed using GraphPad Prism 8 software. Two-tailed Student's t-tests were used to compare differences between two groups. One-way or two-way ANOVA was used to compare three or more groups, and Tukey or Sidak multiple comparison tests were performed as needed. A p-value < 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

[0085] 17. Experimental Results

[0086] (1) Preparation and characterization of BLCDs

[0087] Following the method described in Example 1, *Sanguisorba officinalis* was ground into powder using a herbal grinder. The powder was then reacted in a reaction vessel using a one-step hydrothermal method (200°C, 8 h). The product was dialyzed and freeze-dried under vacuum to obtain carbon dots derived from *Sanguisorba officinalis*, which were named LCDs (results are shown in Figure 1). Figure 1 (A) TEM results showed that the synthesized LCDs were uniform in size, with no obvious aggregation, and the particle size exhibited a Gaussian distribution, with an average particle size of 8.5 ± 0.35 nm (e.g., A). Figure 1 (B in the original text). Further selection of the electron diffraction (SAED) pattern shows that the LCDs exhibit a diffuse ring-shaped diffraction pattern, indicating a disordered state, suggesting that the LCDs surface has an amorphous carbon structure. Figure 2 (A in the text). Meanwhile, XRD data show that LCDs have a broad diffraction peak at 24.5°, corresponding to the (002) crystal plane of graphene, indicating that they possess a highly disordered graphitized structure. Figure 2 (B in the middle).

[0088] To determine the structure and composition of LCDs, we used Raman spectroscopy for preliminary analysis. The data showed that LCDs possess a characteristic Raman peak at 1380 cm⁻¹. -1 The D peak at 1576 cm⁻¹ -1 The G peak at that location indicates the formation of carbon points. The intensity ratio of the D peak to the G peak (I) D / I G The value is 0.74, indicating that LCDs have a graphene-like structure. Figure 2 (C in the text). For quantitative analysis of sp. 2 with sp 3 Based on the relative content of hybrid carbon, we performed baseline correction on the Raman spectrum and peak fitting of the D and G bands. In the peaked spectrum, 1350 cm⁻¹-1 and 1605 cm -1 The peak at that location belongs to sp 2 Carbon, and 1195 cm -1 and 1510 cm -1 The peak at that location belongs to sp 3 Carbon. 2 Carbon and sp 3 The carbon integral intensity ratio is approximately 2.59, and the calculated sp in LCDs is... 2 The relative carbon content is 72.2%, sp 3 The relative carbon content was 27.8%. These results indicate that the graphitization degree of LCDs was 72.2%, while also exhibiting abundant amorphous carbon.

[0089] Subsequently, berberine (BBR) and LCDs were mixed at different mass ratios (1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1) and subjected to hydrothermal reaction in a reactor (100°C, 1 h). The results showed that when the BBR to LCDs ratio was 2:1, the loading efficiency of LCDs on BBR was the highest, reaching 86.1% (…). Figure 2 (D in the original text). After dialysis and vacuum freeze-drying, the synthesized product was named BLCDs. TEM results showed that BLCDs were spherical with a diameter of approximately 72.4 ± 2.1 nm, but with uneven edges and blurred interface contours, indicating that a large amount of BBR may have been grafted onto their surface. Particle size analysis further showed that the particle size of BLCDs in aqueous solution was significantly larger than that of LCDs. Surface potential (Zeta) measurements showed that the surface potential of LCDs was -17.8 mV, BBR was +4.1 mV, and BLCDs was +0.82 mV. These data indicate that LCDs and BBR can self-assemble into supramolecular nanoaggregates, namely BLCDs, through electrostatic interactions.

[0090] Surface functional group analysis of the samples was performed using Fourier transform infrared spectroscopy (FT-IR). Figure 1 (F in the figure). The results show that the characteristic peak of LCDs is located at 3423 cm⁻¹. -1 (OH / NH), 2751cm -1 (CH), 1650cm -1 (C=C / C=O) and 1021cm -1 (CO). However, upon binding with BBR, the OH / NH characteristic peaks of BLCDs red-shift to 3320 cm⁻¹. -1The remaining characteristic peaks showed almost no change, indicating a hydrogen bond interaction between BBR and LCDs. Further UV spectroscopy analysis revealed a broad absorption band in the UV region for LCDs, with a weak absorption peak at 280 nm, which can be attributed to the n→π* transition of oxygen-containing functional groups (such as C=O) on the carbon dot surface. Figure 1 The G in the image shows that BBR exhibits multiple typical absorption peaks at 270 nm, 345 nm, and 430 nm. The absorption spectrum of BLCDs is similar to that of BBR, indicating successful binding between BBR and LCDs. However, the peak positions show a red shift and a hypochromic effect, with absorption peaks located at 290 nm, 365 nm, and 435 nm. The red shift and hypochromic effect of the UV absorption peaks indicate that BBR and LCDs are coupled through hydrogen bonds, forming a new ground-state complex, which also leads to significant quenching of the fluorescence of BLCDs. As can be seen from the images, the powder and solution colors of BLCDs are significantly different from those of LCDs and BBR, and their fluorescence characteristics under UV light also differ, with BLCDs showing significantly weaker fluorescence. Figure 1 The fluorescence spectroscopy data showed that the excitation and emission spectra of BLCDs were different from those of LCDs and BBRs, and the fluorescence intensity was quenched, further confirming that BLCDs are a new ground-state complex formed by the combination of BBR and LCDs. Figure 1 (I in the text). In summary, BBRs and LCDs were successfully assembled to form BLCDs through electrostatic interactions and non-covalent bonding such as hydrogen bonds.

[0091] X-ray photoelectron spectroscopy (XPS) was used to analyze the main components of LCDs and BLCDs. The results showed that the elemental contents of O, N, and C in LCDs were 38.11%, 1.62%, and 60.27%, respectively. Figure 1 (J in the image). Combined with FT-IR data, it can be determined that the surface of LCDs mainly consists of oxygen-containing functional groups, i.e., 3423 cm⁻¹ in the FT-IR spectrum. -1 The characteristic peaks at this location are attributed to OH. Further peak fitting of the C1s spectrum of LCDs revealed characteristic peaks at 284.1 eV, 286.2 eV, and 287.6 eV, corresponding to CN / CO, C=C / CC, and C=O bonds, respectively. A weak NH characteristic peak appeared at 399.5 eV in the N1s spectrum, while a characteristic peak of C-OH / COC appeared at 532.2 eV in the O1s spectrum. These results indicate that the surface of LCDs is rich in oxygen-containing functional groups such as C=O and C-OH.

[0092] After combining with BBR, the elemental contents of O, N, and C in BLCDs are 20.42%, 4.53%, and 75.05%, respectively. Figure 1The nitrogen (N) content in BLCDs is significantly increased and the oxygen (O) content is decreased compared to LCDs, indicating that the bonding of BBR introduces nitrogen-containing groups. The C1s spectra of BLCDs show characteristic peaks at 284.3 eV, 286.1 eV, and 287.2 eV, corresponding to CN / CO, C=C / CC, and C=O bonds, respectively. The N1s spectra show characteristic peaks at 395.8 eV (C=N), 400.8 eV (NH), and 405.8 eV (nitrogen oxides). The O1s spectra show characteristic peaks at 531.3 eV (C-OH / COC) and 532.8 eV (C=O). These results indicate that the bonding of BBR alters the chemical composition of LCDs, significantly increasing the content of nitrogen-containing groups, further confirming the successful assembly of BBR with LCDs.

[0093] (2) Antioxidant and anti-inflammatory properties of BLCDs

[0094] To investigate the role of oxygen-containing functional groups on the surface of LCDs in their SOD-like activity, we first tested the SOD-like activity of LCDs and BLCDs. Figure 3 (A) The results showed that the SOD-like activities of the two were 6.81 × 10⁻⁶. 3 U / mg and 6.64×10 3 U / mg ( Figure 3 The results showed no significant difference in the B content, indicating that the binding of BBR to LCDs does not affect the SOD-like activity of LCDs.

[0095] Subsequently, we passivated the carboxyl and hydroxyl groups using 1,3-propanesulfonic acid lactone (PS). PS reacts with the carboxyl and hydroxyl groups on the carbon dot surface to form ester and ether bonds, respectively, yielding the modified product LCDs-PS. Under acidic conditions, the ester bonds are hydrolyzed, while the ether bonds are not. Therefore, after hydrolyzing LCDs-PS in 0.1 M HCl solution, we obtained LCDs with only the hydroxyl groups passivated, named LCDs-PS-HCl. Activity assays showed that the SOD-like activity of LCDs-PS decreased to 1.28 × 10⁻⁶. 3 U / mg indicates that passivation of hydroxyl and carboxyl groups significantly reduces the SOD-like activity of LCDs. Figure 3 (B) For LCDs-PS-HCl, the carboxyl groups are restored while the hydroxyl groups remain passivated, and the SOD-like activity recovers to 2.73 × 10⁻⁶. 3 U / mg, but failed to fully recover to the original level ( Figure 3 (B in the text). This indicates that hydroxyl groups also play a crucial role in the SOD-like activity of LCDs.

[0096] To further investigate the role of carbonyl groups in the SOD-like activity of LCDs, we reduced the carbonyl groups on the LCD surface using NaBH4. The SOD-like activity of the resulting LCDs-NaBH4 significantly decreased, dropping to 0.74 × 10⁻⁶. 3 U / mg indicates that the carbonyl group plays an important role in the SOD-like activity of LCDs. Figure 3 (B in the original text). To verify this conclusion, we subsequently treated with HNO3 to promote the regeneration of carbonyl groups, and the results showed that the SOD-like activity of LCDs-NaBH4-HNO3 was significantly restored to 3.12 × 10⁻⁶. 3 U / mg ( Figure 3 The presence of B in the figure further confirms the contribution of the carbonyl group to the SOD-like activity of LCDs. In summary, the above results indicate that oxygen-containing functional groups such as carboxyl, hydroxyl, and carbonyl groups are key factors for LCDs to exert SOD-like activity.

[0097] We measured the scavenging efficiency of LCDs and BLCDs for total reactive oxygen species (ROS) at different time points in vitro. Figure 3 The results showed that both LCDs and BLCDs significantly scavenged ROS over time, with no significant difference between them. This result is similar to the SOD-like activity experiment, indicating that BBR binding does not affect the ROS scavenging activity of LCDs. Figure 3 B and Figure 3 (C in the text). To further investigate the SOD-like activity of LCDs and BLCDs, we tested the effects of different concentrations of LCDs and BLCDs on •OH and •O2 at 24 h. - The in vitro scavenging efficiency was assessed. Results showed that both LCDs and BLCDs significantly scavenged •OH and •O2 in a concentration-dependent manner. - Furthermore, there was no significant difference between the two, further confirming the SOD-like activity of LCDs and BLCDs. Figure 3 (D and 3E). In summary, BLCDs inherit the SOD-like activity of LCDs, and can interact with •OH and •O2 through oxygen-containing functional groups such as carboxyl, hydroxyl, and carbonyl groups on their surface. - It exhibits significant ROS scavenging capabilities.

[0098] BLCDs not only possess the nanoscale characteristics of LCDs but also load berberine (BBR), which has significant anti-inflammatory activity. As a multi-target drug, BBR can effectively reduce oxidative stress levels in the body by regulating multiple signaling pathways (such as NF-κB, Nrf2, EIF2AK2, JAK / STAT, AKT, and NLRP3 pathways) and inhibiting the expression of pro-inflammatory cytokines (such as TNFα, IL6, IL1α, and IL1β). Figure 3 We first constructed an in vitro enteritis cell model using LPS-induced NCM460 cells, and then treated them with LCDs, BBR, and BLCDs (50 μg / mL), respectively. The results showed that, unlike the in vitro ROS scavenging results, the antioxidant capacity of BLCDs was significantly superior to that of LCDs (F in the original text). Figure 3 The G in this context is attributed to the fact that BBR exerts an indirect antioxidant effect within cells through its anti-inflammatory mechanism, while BLCDs combine the SOD-like activity of LCDs with the natural anti-inflammatory activity of BBR. The synergistic effect of these two factors results in a more significant reactive oxygen species scavenging capacity within inflammatory cells. Figure 3 (F in the middle).

[0099] In fact, in terms of intracellular antioxidant activity, LCDs directly scavenged ROS more efficiently than BBR indirectly inhibited it. Regarding anti-inflammation, we found that BBR significantly inhibited the expression of pro-inflammatory factors, substantially downregulating the expression of IL6, TNFα, IL1α, and IL1β at both mRNA and protein levels. This is closely related to its multi-target regulated natural anti-inflammatory pharmacological effects. Figure 3 (I in 3 and J in 3). It is noteworthy that BLCDs also inherit the natural anti-inflammatory effects of BBR, and can also achieve synergistic regulation of inflammation through the antioxidant mechanism of LCDs. Compared with using LCDs or BBR alone, BLCDs have a more significant regulatory effect on pro-inflammatory factors, restoring LPS-induced intracellular inflammation levels in NCM460 cells to levels close to those in the PBS group (…). Figure 3 (I and J in 3). Under the synergistic effect of BBR's anti-inflammatory effect and LCDs' antioxidant effect, BLCDs significantly rescued the viability of LPS-induced NCM460 cells ( Figure 3 The K in 3 and the L in 3). Furthermore, different concentrations (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μg / mL) of BLCDs showed no significant toxicity to NCM460 cells, indicating good biocompatibility. Figure 4 (A in 4 and B in 4). In summary, BLCDs combine the SOD-like activity of LCDs with the natural anti-inflammatory ability of BBR, making them a nanomedicine with significant research value.

[0100] (3) RNA-seq suggests the mechanism of BLCDs in treating UC.

[0101] Mechanistic analysis of LPS-induced NCM460 cells before and after BLCDs treatment using RNA-seq revealed that IL10 was the most significantly upregulated gene after BLCDs treatment (approximately 11.5-fold), and the IL10 production pathway was significantly enriched. Figure 5 (A~D in the original text). Transcriptome radar plots showed that although IL10 expression slightly increased under LPS induction, it was significantly upregulated after BLCDs intervention. Figure 6 (A in the original text). We further examined the expression levels of IL10 mRNA and protein, validating the RNA-seq results (…). Figure 6 In addition, in this pathway, LPS induction significantly upregulated NLRP3, a downstream target molecule of IL10 (7.6-fold), while BLCDs treatment significantly decreased NLRP3 expression (B). Figure 6 (A) NLRP3 mRNA and protein level assays confirmed that BLCDs could significantly reverse LPS-induced NLRP3 elevation (…). Figure 6 (C in the text). Simultaneously, we also examined the expression of downstream pro-inflammatory cytokines IL1β and IL18 regulated by NLRP3. The results showed that LPS-induced IL1β and IL18 expression significantly decreased after BLCDs intervention, further supporting the regulation of NLRP3 expression by BLCDs through the IL10 pathway. Figure 6 The increase in IL10 and the decrease in NLRP3 predict a reduction in cellular inflammation levels. We examined the expression of classic inflammatory factors TNFα and IL6, confirming that BLCDs significantly alleviated inflammation. Figure 6 (E in the text).

[0102] To further demonstrate that BLCDs exert their anti-inflammatory and antioxidant functions in vivo through IL10, we conducted an IL10 inhibition experiment on RAW264.7 cells, which are more sensitive to inflammation regulation. LPS-induced RAW264.7 cells were co-treated with BLCDs and JES5-2A5 (an IL10 inhibitor, MCE, HY-P990001). The results showed that the expression level of the anti-inflammatory factor IL10 was significantly inhibited. Figure 7 In A), downstream NLRP3 and its effector pro-inflammatory factors IL1β and IL18 were significantly upregulated ( Figure 7 (B in B and C in 7), thus failing to alleviate cellular inflammation levels (TNFα and IL6 were highly expressed) Figure 7(D in the original text). This result indicates that BLCDs exert a key anti-inflammatory effect through the regulation of NLRP3 and its downstream pro-inflammatory factors by IL-10. Furthermore, fluorescence intensity detection of cellular reactive oxygen species also showed that BLCDs exert their antioxidant effect through IL-10, and that inhibiting IL-10 significantly weakens the ability of BLCDs to rescue inflammatory cells. Figure 7 (E in 7 and F in 7). In summary, BLCDs, which possess both anti-inflammatory and antioxidant functions, mainly regulate IL10 in inflammatory cells.

[0103] (4) Evaluation of the efficacy of BLCDs in UC

[0104] To evaluate the in vivo therapeutic effect of BLCDs on inflammatory bowel disease, we established a mouse model of ulcerative colitis (UC) using 3% DSS in a free-drinking environment. On days 7, 9, and 11 after modeling, UC mice were treated with 5-ASA (positive control), LCDs, BBR, a mixture of BBR and LCDs (BBR+LCDs), and BLCDs, respectively. Before formal treatment, a dose-response experiment was conducted to determine the optimal dosage of BLCDs. The results showed that 80 mg / kg of BLCDs achieved the best DAI score; therefore, this dosage was selected for subsequent experiments. Figure 8 Healthy mice treated with PBS served as controls, while DSS-induced UC mice served as the model group. Mice were sacrificed on day 14, and major organs were collected for subsequent analysis. Figure 9 (A in the middle).

[0105] Anatomical diagrams of the mouse colon showed that the DSS model group had the shortest colon length, while the BLCDs treatment group had the longest colon length, approaching the level of the PBS healthy control group. Figure 9 (B in the text). Pre- and post-treatment anal photography records showed that mice in the DSS model group exhibited obvious "wet tail" phenomenon (perianal dampness caused by diarrhea). All treatment groups showed varying degrees of improvement, with the BLCDs group showing the most significant effect, essentially recovering to normal levels. Figure 9 (C) The BBR+LCDs group showed the second-best improvement, suggesting that simple mixed administration is less effective than the integrated BLCDs nanomedicine in improving UC, further indicating that BLCDs, as a whole nano-formulation, can exert its anti-inflammatory effect more precisely and efficiently. Figure 9 (B in 9 and C in 9). Mouse weight changes, DAI scores, and colon length measurements further confirmed the above observations. Figure 9 The DF in the middle). It is worth noting that BLCDs were superior to the positive drug 5-ASA and its component LCDs and BBR alone in restoring mouse body weight, improving DAI scores and colon length. Figure 9(DF in the data). These data not only confirm the in vivo therapeutic safety of BLCDs, but also highlight the synergistic advantage of BLCDs in the treatment of UC, combining the antioxidant properties of LCDs with the natural anti-inflammatory activity of BBR.

[0106] To further investigate the therapeutic effect of BLCDs on UC, we performed pathological evaluation, goblet cell (secreting mucin, maintaining the intestinal mucosal barrier) count, and intestinal fibrosis analysis on the colonic tissue of mice in each group. The results showed that the colonic pathological score, goblet cell positive count, and fibrosis area of ​​the BLCDs-treated group were closest to those of the PBS healthy control group, and the therapeutic effect was the most significant among all treatment groups. Figure 9 In addition, we examined the expression levels of ZO-1 and Occludin, key molecular markers reflecting intestinal tight junction integrity, permeability, and barrier function. The results showed that BLCDs significantly reversed DSS-induced intestinal barrier dysfunction, substantially upregulated the expression of ZO-1 and Occludin, and were superior to other treatment groups (GL). Figure 10 A and Figure 9 (B in the text). Intestinal oxidative stress level detection showed that the DHE fluorescence intensity was significantly reduced in the BLCDs group, indicating that BLCDs can effectively inhibit intestinal oxidative stress levels in UC mice. Figure 10 C and Figure 10 The results, consistent with in vitro cell experiments, indicate that surface BLCDs not only inherit the SOD-like activity of LCDs, but also, the loaded BBR can inhibit reactive oxygen species generation through anti-inflammatory pathways, thus exhibiting a stronger ROS scavenging capacity in vivo than that of single components. Figure 10 C and Figure 10 (D in the text). Furthermore, H&E staining results showed that BLCDs did not cause significant toxic damage to the major organs (heart, liver, spleen, lung, and kidney) of mice during treatment. Figure 11 ).

[0107] To verify whether the in vivo anti-inflammatory and antioxidant molecular mechanism of BLCDs is consistent with the results of RNA-seq and in vitro cell experiments, we detected the expression levels of IL10 and its downstream target molecules NLRP3, as well as the pro-inflammatory cytokines IL1β and IL18, in the colon tissue of mice in each group. The results showed that BLCDs significantly upregulated the expression of IL10 in DSS-induced mice, thereby inhibiting the expression levels of its downstream target gene NLRP3 and the pro-inflammatory cytokines IL1β and IL18. Figure 10 The F in F and the G in G10). The improvement of the in vivo inflammatory microenvironment made the inflammation level of DSS-induced mice close to that of the PBS group, basically restoring them to a normal physiological state. Figure 10 (H in the text).

[0108] Near-infrared fluorescence imaging results showed that LCDs and BLCDs were mainly enriched in intestinal tissue after oral administration. Figure 10 I). BLCDs reached peak intestinal accumulation 3 hours after administration and remained accumulated after 24 hours. LCDs, on the other hand, reached peak accumulation within 1 hour, and their metabolic clearance in the intestine was significantly faster than that of BLCDs. This is because the particle size of BLCDs (approximately 70 nm) is significantly larger than that of LCDs (8.5 nm), making them more likely to remain in intestinal tissue, potentially resulting in a more sustained and potent therapeutic effect. Within the 24–72 hour time window, the distribution of both BLCDs and LCDs in the intestine gradually decreased, with some being excreted through feces and urine. Figure 10 J in the middle.

[0109] In summary, BLCDs can remain in the intestine for a long time, thereby synergistically exerting the antioxidant properties of LCDs and the anti-inflammatory activity of BBR. Together, they upregulate IL10 expression, inhibit NLRP3 production and pro-inflammatory factor secretion, effectively alleviate DSS-induced intestinal oxidative stress in mice, maintain the integrity and function of the intestinal barrier, and thus show significant therapeutic effects on UC.

[0110] (5) Effects of BLCDs on gut microbiota and metabolites in UC

[0111] Changes in gut microbiota are an important indicator for evaluating the treatment efficacy of ulcerative colitis (UC). To further explore the impact of BLCDs on gut microbiota, we performed 16S rDNA sequencing on fecal samples from mice in each group. Sequencing data analysis showed that BLCDs intervention significantly increased the abundance (observed OTUs), diversity (Shannon curve), and richness (rarefaction curve) of gut microbiota. Non-metric multidimensional scaling (NMDS) analysis based on microbial community composition showed that the gut microbiota of the BLCDs group was significantly different from that of the DSS model group and other treatment groups. Figure 12 (AD in the text).

[0112] The relative abundance of gut microbiota and LDA scores showed that after BLCDs treatment, beneficial bacteria such as *Ligilactobacillus*, *Lactobacillus*, and *Akkermansia* significantly increased, while harmful bacteria such as *Escherichia-Shigella* and *Klebsiella* significantly decreased. Notably, BLCDs exhibited a significantly better positive regulatory effect on gut microbiota than the positive control drug 5-ASA (mesalazine). Figure 12 F and Figure 12 (G in the middle).

[0113] Changes in the gut microbiota can further affect the composition of gut metabolites, thereby regulating the progression of UC. We examined the gut metabolites of mice in the BLCDs and DSS groups and found significant differences in the metabolite profiles between the two groups. Figure 13 (A) Intestinal metabolite heatmaps showed that after BLCDs treatment, the levels of trimethylamine oxide (TMAO) in the intestines of all mice were significantly reduced, while those in the DSS group were significantly increased. Figure 13 (B in the original text). TMAO is a key molecule in gut microbial metabolism, mainly produced by the oxidation of trimethylamine (TMA), and can be distributed to various organs throughout the body via the bloodstream. Elevated TMAO levels induce intestinal inflammation, oxidative stress, and DNA damage, promoting the progression of intestinal lesions to precancerous states. Studies have shown that the gut microbe Klebsiella is involved in the metabolic process of TMA, which is consistent with our observation of reduced Klebsiella abundance in the BLCDs group. Figure 12 Therefore, we further examined the levels of TMA and TMAO in feces and plasma. The results showed that after BLCDs treatment, both TMA and TMAO in feces and plasma were significantly downregulated (G). Figure 13 C and Figure 13 (D in the text). The above results indicate that BLCDs intervention can improve the therapeutic effect of UC by regulating the gut microbiota and thus altering the expression of the DSS-induced intestinal metabolite TMAO in mice.

[0114] To verify the regulatory effect of BLCDs on TMAO, we conducted our study in normal intestinal organoids (CNOs). The results showed that TMAO treatment significantly reduced the diameter and activity of CNOs, similar to LPS treatment, and induced significant inflammatory lesions. Figure 13 The E in the text). BLCDs treatment, regardless of whether LPS or TMAO-induced organoids are affected, can significantly reverse the decrease in diameter and viability, reduce oxidative stress levels, and alleviate inflammatory lesions. Figure 14 (AC in the middle). We also examined the expression of IL10 and its downstream gene NLRP3 and effector pro-inflammatory factors IL1β and IL18. The results showed that TMAO stimulation significantly upregulated the expression of NLRP3 and pro-inflammatory factors, indicating that TMAO is a key metabolite that exacerbates intestinal inflammation. Figure 13 F in Figure 13 H and Figure 13 More importantly, BLCDs treatment significantly induced the production of IL10 in LPS / TMAO-induced CNOs (I). Figure 13In G), downregulating the expression of NLRP3 and effector pro-inflammatory factors IL1β and IL18 ultimately improves the inflammatory microenvironment of CNOs (decreased expression of TNFα and IL6). Figure 13 J in the middle.

[0115] To fully verify that TMAO-mediated intestinal inflammation can indeed be blocked by IL10 regulated by BLCDs, we conducted an IL10 inhibition experiment. BLCDs and AS101 (an IL10 inhibitor, MCE, HY-101019) were co-incubated with TMAO-induced CNOs, and the results showed that the therapeutic effect of BLCDs was significantly reversed. Figure 15 (A in 15 and B in 15). Under AS101 intervention, the effect of BLCDs in promoting IL-10 production was inhibited, resulting in the inability to restore the diameter and activity of TMAO-induced CNOs, and maintaining a high level of oxidative stress. Figure 15 (CF in the middle).

[0116] In summary, BLCDs can regulate the abundance of gut microbiota in UC mice and significantly reduce the production of the intestinal inflammation-related metabolite TMAO. Furthermore, BLCDs can inhibit TMAO-mediated inflammatory responses through the IL-10 production pathway, thereby effectively improving the progression of UC.

[0117] (6) In vivo safety experiments of BLCDs

[0118] Finally, we conducted a systematic evaluation of the in vivo safety of BLCDs. Healthy mice treated with PBS served as controls; major organs and blood samples were collected from the mice on day 7 for analysis. Figure 16 (A in the text). Simultaneously, we designed a long-term safety observation experiment, and the results showed no significant difference in body weight change between the BLCDs group and the PBS group over 60 days (…). Figure 16 B in the text). H&E staining results showed that BLCDs treatment did not cause significant toxic damage to the heart, liver, spleen, lungs, kidneys, and intestines of mice. Figure 16 (C) Blood routine analysis showed that BLCDs administration did not cause abnormal changes in the number of white blood cells (WBC), platelets (PLT), hemoglobin (HGB), and red blood cells (RBC). Figure 16 (D in the text). Serum biochemical indicators further showed that BLCDs had no significant effect on liver and kidney function in mice. Figure 16 (E in the text). Furthermore, in vitro hemolysis experiments confirmed that BLCDs have no hemolytic toxicity to mouse erythrocytes. In summary, BLCDs exhibit good in vivo safety (E in the text). Figure 16 (F in the middle).

[0119] 18. Conclusion

[0120] This invention successfully constructed a nanocomposite BLCDs formed by the self-assembly of LCDs and berberine. This composite forms a stable supramolecular structure through electrostatic interactions and hydrogen bonding, possessing both the SOD-like antioxidant activity of LCDs (dependent on surface carboxyl, hydroxyl, and carbonyl groups) and the multi-target anti-inflammatory function of berberine. In vitro and in vivo experiments showed that BLCDs exert their therapeutic effect through a dual synergistic mechanism: directly scavenging reactive oxygen species through surface oxygen-containing functional groups, while simultaneously inhibiting the NLRP3 inflammatory pathway and downstream pro-inflammatory factors by promoting IL-10 expression. In a DSS-induced ulcerative colitis mouse model, BLCDs showed superior efficacy compared to the positive control drug 5-ASA and its single component, significantly improving intestinal inflammation, oxidative stress, and barrier dysfunction. Its therapeutic advantage stems from the intestinal retention effect conferred by its larger particle size, as well as its regulatory effects on intestinal flora and metabolites, particularly by inhibiting Klebsiella to reduce TMAO production, thereby activating the multi-cascade regulatory network of IL-10. Safety assessments showed that BLCDs have good biocompatibility and clinical translational potential. This invention provides a new scientific perspective for understanding the compatibility mechanism of traditional Chinese medicine compound prescriptions, and also provides a research paradigm that can be referenced for the development of nanomedicines based on the active components of traditional Chinese medicine.

Claims

1. Application of herbal carbon dots loaded with berberine in the preparation of drugs for treating ulcerative colitis.

2. The application according to claim 1, characterized in that, The herbal carbon dots loaded with berberine were obtained by self-assembling Rehmannia glutinosa carbon dots with berberine.

3. The application according to claim 2, characterized in that, The mass ratio of the carbon dots of *Rehmannia glutinosa* to berberine is 1:5 to 5:

1.

4. The application according to claim 2, characterized in that, The carbon dots of *Rehmannia glutinosa* are prepared by a one-step hydrothermal method. Preferably, the one-step hydrothermal method includes the following steps: adding *Rehmannia glutinosa* powder and pure water in a mass-to-volume ratio of 1:1 to 1:10 g / mL into a reaction vessel and reacting at 150 to 300°C for 4 to 12 hours; after the reaction, filtering, dialysis and vacuum freeze-drying are performed to obtain the carbon dots of *Rehmannia glutinosa*.

5. The application according to any one of claims 2 to 4, characterized in that, The average particle size of the carbon dots in the raw *Ulmus pumila* is 8.5 ± 0.35 nm. Preferably, the concentration of the carbon dots in the raw *Ulmus pumila* is 1~100 µg / mL.

6. The application according to claim 1, characterized in that, The dosage forms of the drug include liquid preparations, tablets, capsules, or dry suspensions.

7. The application according to claim 1, characterized in that, The drug is one that upregulates IL10 gene expression and / or inhibits the expression of the NLRP3 inflammasome pathway and its downstream pro-inflammatory factors.

8. The application according to claim 1, characterized in that, The drug is used to improve intestinal inflammation, oxidative stress, and barrier dysfunction.

9. The application according to claim 1, characterized in that, The drug is a drug that enhances the abundance and diversity of intestinal flora, and / or inhibits the growth of harmful bacteria such as Klebsiella spp., and / or reduces the formation of trimethylamine oxide.

10. The application according to any one of claims 2 to 5, characterized in that, The dosage of the carbonized Rehmannia glutinosa was 1-100 mg / kg mice.

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