Calcined lotus leaf-derived carbon dots: enhanced hemostasis, anti-inflammatory, immunomodulatory properties for ulcerative colitis management

Yu Zheng; Jiangcui Liu; Ying Ma; Yunyu Zhang; Heng Liu; Rui Chen

doi:10.1186/s12951-026-04208-5

. 2026 Mar 1;24:319. doi: 10.1186/s12951-026-04208-5

Calcined lotus leaf-derived carbon dots: enhanced hemostasis, anti-inflammatory, immunomodulatory properties for ulcerative colitis management

Yu Zheng ^1,^#, Jiangcui Liu ^2,^#, Ying Ma ¹, Yunyu Zhang ^1,³, Heng Liu ^2,^✉, Rui Chen ^1,^3,^✉

PMCID: PMC13059337 PMID: 41765900

Abstract

Ulcerative Colitis (UC) is a chronic condition characterized by damage to the intestinal mucosal barrier, resulting in bleeding, increased oxidative stress, persistent inflammation, and immune dysregulation. Lotus Leaf (LL), recognized for its dual role as both food and medicine, has demonstrated significant antioxidant and anti-inflammatory properties. Furthermore, its calcined derivative, Lotus Leaf Charcoal (LLC), enhances its astringent, hemostatic, and antidiarrheal effects, positioning it as a promising candidate for the management of UC in both dietary and medicinal contexts. This study aims to explore the potential of LLC in the treatment of UC and its material basis. LLC was prepared by simulating traditional calcination processes through high-temperature pyrolysis at 450 °C, and it was found to contain a large number of spherical nanoparticles uniformly distributed in the range of 0.5–3 nm, exhibiting good dispersibility and stability. In vitro and in vivo experiments demonstrate that LLC exhibits a dose-dependent hemostatic effect which significantly increases platelet (PLT) count, elevates fibrinogen (FIB) concentration, and shortens activated partial thromboplastin time (APTT) and thrombin time (TT). Additionally, LLC shows excellent free radical scavenging abilities against DPPH•, ABTS+•, •OH, and O2-• radicals. Furthermore, LLC exhibits remarkable gastrointestinal stability and long-term retention. In the dextran sulfate sodium (DSS)-induced mouse model of UC, LLC significantly alleviates weight loss, reduces the disease activity index (DAI) and colonic mucosal injury index (CMDI), improves colonic shortening and tissue pathological damage. It downregulates the levels of pro-inflammatory factors such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), decreases indicators of oxidative stress like reactive oxygen species (ROS) and malondialdehyde (MDA), and may inhibit pyroptosis of colonic epithelial cells by suppressing the excessive activation of the NLRP3/Caspase-1/GSDMD signaling pathway. Additionally, it modulates the ratio of CD4+/CD8+ T cells and the Th17/Treg balance in the spleen, thereby restoring immune homeostasis. Additionally, LLC upregulates the expression of tight junction proteins Claudin-1 and Occludin, promoting intestinal barrier repair, and increases the abundance of beneficial bacteria while inhibiting the proliferation of harmful bacteria, ultimately reshaping the intestinal microbiota structure. In summary, LLC contains a substantial amount of carbon nanodots, which improve UC through multiple mechanisms, including mucosal repair, hemostasis, antioxidant effects, anti-inflammatory actions, pyroptosis inhibition, immune modulation, and microbiota regulation. These findings provide a preclinical foundation for developing carbon-based therapeutics for UC.

Graphical Abstract

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Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-026-04208-5.

Introduction

Ulcerative colitis (UC), as an idiopathic chronic inflammatory bowel disease (IBD), is characterized by persistent inflammation of the rectal and colonic mucosa as its core pathological feature. It is often accompanied by oxidative stress, dysregulated immune responses, and impaired intestinal barrier function. Clinically, it presents with bloody diarrhea, mucosal ulcers, and alternating patterns of exacerbation and remission [1–3]. The cure rate for this disease is low, and it is prone to recurrence. Prolonged inflammatory stimulation may also progress to colon cancer, and it has been classified as one of the modern refractory diseases by the World Health Organization [4]. Currently, the main therapeutic agents for UC include 5-aminosalicylic acid, glucocorticoids, and immunosuppressants [5, 6]. However, the existing therapies have limited effectiveness in alleviating symptoms and controlling relapses, and they are often accompanied by adverse reactions. In-depth research has revealed that patients with UC experience an issue of excessive production of reactive oxygen species (ROS) in their intestinal mucosa [7]. The accumulation of ROS not only directly damages the integrity of intestinal microvilli but also exacerbates the "ROS-inflammation" vicious cycle, triggering pathological inflammatory responses [8, 9]. Excessive ROS can serve as terminal electron acceptors for facultative anaerobes during anaerobic respiration, promoting the proliferation of harmful bacteria and leading to dysbiosis of the intestinal ecosystem [10]. Therefore, there is an urgent need to find new therapeutic approaches that can effectively eliminate ROS, regulate immune responses, and repair intestinal barriers. Traditional Chinese medicine, particularly charcoal-based remedies, has shown significant potential in the treatment of UC due to its astringent, hemostatic, and antidiarrheal properties. Modern research indicates that high-temperature carbonization can endow traditional Chinese medicine with enhanced hemostatic and antidiarrheal effects [11]. As a result, carbonized medicines are widely used in the treatment of hemorrhagic diseases and gastrointestinal disorders [12]. For instance, the raw product of Rhizoma Rhei, characterized by its bitter and cold properties, exhibits a purgative effect. When processed into charcoal, it demonstrates an anti-diarrheal effect by inhibiting intestinal motility, regulating intestinal flora, protecting the intestinal mucosa, and reducing secretion [13]. Moreover, the efficacy of honeysuckle charcoal in ameliorating gastric tissue bleeding and gastric ulcers in a rat model of blood-heat bleeding is showed greater improvement than the raw product [14]. Additionally, Tangerine Peel Charcoal can exert its antidiarrheal effect by reducing serum serotonin levels, thereby decreasing intestinal motility in diarrheic rats [15].

Lotus leaf (LL) is the dried leaf of the plant Nelumbo nucifera Gaertn., which belongs to the family Nymphaeaceae. As a resource utilized for both medicinal and dietary purposes [16], fresh LL is known for its effects in clearing heat and eliminating dampness, and cooling the blood to stop bleeding [17]. It is widely employed in clinical practice to treat symptoms such as diarrhea, vomiting, hematochezia, and uterine bleeding [18]. Modern research has confirmed that LL contain flavonoids, alkaloids, and other components, which exhibit antioxidant, anti-inflammatory, and neuroprotective effects [19]. Lotus leaf charcoal (LLC) are products derived from the high-temperature calcination of LL under closed hypoxic conditions, which significantly enhance their astringent and hemostatic properties following carbonization. LLC is primarily utilized to address various bleeding symptoms, including hemoptysis, epistaxis, hematochezia, and metrorrhagia. Research indicates that it can improve hemostatic effects by influencing both the endogenous and exogenous coagulation pathways, as well as the fibrinolytic system in rat models [20]. The carbonization process significantly alters the composition of medicinal materials. For instance, after carbonizing the lotus pod, the isoquercitrin content decreases to zero, while the quercetin content shows a reduction of 82.3% [21]. Research indicates that no components were detected by HPLC in LL roasted at 300 °C [22], whereas traditional roasting temperatures typically exceed 300 °C. Although the original active components, such as alkaloids and hyperoside, in LLC significantly decrease or even disappear with prolonged carbonization time, its hemostatic efficacy is stronger than that of fresh LL. This suggests that new substances with astringent and hemostatic properties may have been generated in LLC.

With the advancement of nanomaterials technology, researchers have increasingly recognized that the material basis of carbon-based drugs may be linked to their nanostructured components. In medicinal charcoals such as Typha angustifolia charcoal [23], Sparganium stoloniferum charcoal [24], Lonicera japonica charcoal [25], and Sophora flavescens charcoal [26], charcoal-based nanoparticles have been confirmed as the key material basis for their pharmacological effects [27]. In-depth research has revealed that carbon-based nanoparticles possess unique advantages in the treatment of gastrointestinal diseases. Carbon dots derived from Atractylodes macrocephala can alleviate inflammation and oxidative stress by modulating the NF-κB/NLRP3 axis, while also regulating the diversity and composition of gut microbiota to improve gastric ulcers [28]. The carbon-based nanocomponents of Sophora flavescens can reduce the levels of NF-κB and the concentrations of inflammatory cytokines, inhibit gastric tissue inflammation, and alleviate alcohol-induced oxidative stress to improve acute gastric ulcers [26]. Additionally, the carbon-based nanomaterials from Senna alexandrina also demonstrate an antagonistic effect on the hypermotility of the small intestine, thus serving as an antidiarrheal agent [29]. These studies indicate that the nano-components of carbon-based drugs can exert therapeutic effects by modulating inflammation, oxidative stress, and gut microbiota across multiple pathways. As a traditional charcoal medicine, LLC has been used since ancient times for various bleeding conditions such as hemoptysis, hematemesis, hematuria, metrorrhagia, and postpartum hemorrhage [18]. However, its research should not be limited to hemostatic effects. Considering the pathological characteristics of UC, which include inflammation exacerbation, oxidative stress imbalance, intestinal barrier damage, and dysbiosis, as well as the multi-target action characteristics of carbon-based nanomaterials, the potential of LLC in immune regulation, anti-inflammation, and antioxidation merits further exploration.

Based on this, the present study employs a high-temperature pyrolysis method to prepare LLC, and stable, homogeneous carbon-based nanoparticles have been discovered within its system. LLC exhibits excellent in vitro hemostatic activity and ROS scavenging ability. In a dextran sulfate sodium (DSS)-induced mouse model of UC, the therapeutic effect of LLC is significantly superior to that of LL. Further experiments confirm that LLC can improve UC through multiple pathways, including accelerating hemostasis, regulating inflammatory responses and oxidative stress, immune modulation, and repairing colonic barrier damage (Scheme 1). Additionally, this study employed metagenomic sequencing technology to explore the potential mechanisms by which LLC exerts its therapeutic effects. In summary, this research suggests that LLC has the potential to become a natural drug with multiple pharmacological activities, providing new research directions and strategies for the treatment of complex diseases such as UC.

Scheme 1 — Schematic representation of the preparation of LL and LLC and their oral administration to improve the colonic microenvironment in mice with UC

Materials and methods

Materials

LL (batch number: 24070111) were purchased from Kangmei Pharmaceutical Co., Ltd. (Shenzhen, China). DSS (M.W: 36,000–50000, batch number: M0529D) was obtained from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Mesalazine (batch number: 11032–206) was acquired from Shenzhen Lijing Biochemical Technology Co., Ltd. (Shenzhen, China). The fecal occult blood test kits were purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd. (Nanjing, China). The dialysis membrane (MWCO: 1 kDa) was obtained from Rida Henghui Technology Development Co., Ltd. (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits for tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) were sourced from Thermo Fisher Scientific Inc. (Hangzhou, China), while the ELISA kit for interferon-gamma (IFN-γ) was purchased from Sewei Biotechnology Co., Ltd. (Wuhan, China). The ELISA kits for interleukin-17 (IL-17) and interferon-gamma (IFN-γ) were obtained from LianKe Biotechnology Co., Ltd. (Hangzhou, China). The BCA protein quantification kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Lipopolysaccharide (LPS) was obtained from Sigma-Aldrich (Burlington, Massachusetts, USA). The dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Nanjing Senbeijia Biotechnology Co., Ltd (Nanjing, Jiangsu, China). Primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The total RNA extraction kit (BS258A) was obtained from LanJieKe Biotechnology Co., Ltd. (Beijing, China). The All-in-One First-Strand Synthesis MasterMix (with dsDNase, EG15133S) and Taq SYBR^® Green qPCR Premix (Universal, EG20117M) were purchased from YuGong BioTech Co., Ltd. (Taizhou, China). All experiments were conducted using deionized water (DW).

Preparation of LL and LLC extracts

90 g of LL herbal medicine was crushed and subsequently decocted twice with 30 times the volume of deionized water at a temperature of 100 °C for 2 h. The resulting solution was concentrated and freeze-dried to obtain the LL extract.

90 g of LL herbal medicine was placed in a crucible, sealed with aluminum foil, and then subjected to calcination in a muffle furnace at 350 °C, 450 °C and 550 °C for 1 h [30], yielding LLC. After crushing the LLC, it was decocted twice with deionized water at a ratio of 30 times the weight of LLC, under the condition of 100 °C for 2 h each time. The resulting solution was concentrated and freeze-dried to obtain the LLC extract.

After preparation, the LL and LLC extracts were separately diluted with physiological saline to appropriate concentrations for subsequent pharmacological intervention in animal experiments.

Structural characterization

The morphology and size distribution of the LLC extract were observed using high-resolution transmission electron microscopy (HRTEM, Jem-2100F, 200 kV, Tokyo, Japan). The polymer dispersion index (PDI) and Zeta potential were measured using a Malvern particle size analyzer (Malvern Zetasizer Nano ZS 90, Malvern Panalytical, Malvern, UK). The absorption spectra were obtained using a UV–visible (UV–vis) spectrophotometer (Cecil CE 2021, Cecil Instruments Ltd., Cambridge, UK). A Fourier-transform infrared (FT-IR) spectrometer (Nicolet iS20, Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the surface functional groups of the LLC extracts in the range of 600 to 4000 cm⁻¹. Proton nuclear magnetic resonance (¹H NMR) spectra were recorded using a nuclear magnetic resonance spectrometer (AVANCE NEO 400 MHz, Bruker, Fällanden, Switzerland) with D₂O as the solvent. Fluorescence (FL) spectra were obtained using a fluorescence spectrophotometer (LS-45, PerkinElmer, Yokohama, Japan). X-ray diffraction (XRD) was employed to characterize the crystal structure using a diffractometer (Bruker D8 Advance, Karlsruhe, Germany). Raman spectroscopy tests were conducted with a confocal Raman spectrometer (LabRAM HR Evolution, Horiba Scientific, Kyoto, Japan), with an excitation light wavelength of 532 nm. The elemental composition and elemental analysis of the LLC extract were performed using X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific K-Alpha, Waltham, MA, USA), utilizing a monochromatic Al Kα 150 W X-ray source, with a full scan pass energy of 200 eV and a high-resolution scan pass energy of 30 eV. The surface morphology and elemental mapping of the LLC were investigated using a field emission scanning electron microscope (FE-SEM, Hitachi SU8600, Tokyo, Japan) equipped with an energy-dispersive X-ray spectroscopy (EDS) system (Oxford Ultimax 40, Oxford Instruments, UK). The photoluminescence (PL) lifetime was measured by a steady-state and transient fluorescence spectrometer (FLS-1000, Edinburgh Instruments, Livingston, UK).

Calculation of the carbonization yield (CY)

The carbonation yield (CY) of LLC was determined by gravimetric method. In short, an accurate mass of completely dried LL precursor is weighed and placed in a crucible. Then the pyrolysis process is carried out at the specified temperature and duration. After the system was naturally cooled to room temperature, the obtained black product (LLC) was collected and accurately weighed. The CY is calculated according to the following equation:

where CY denotes the carbonization yield of the test sample; m_LL denotes the mass of the initial precursor, and m_LLC denotes the mass of carbon powder obtained after pyrolysis. The reported values represent the average of at least three independent parallel experiments.

Measurement of the quantum yield (QY)

The quantum yield (QY) of LLC was determined at room temperature using the comparative method [31] with quinine sulfate in 0.1 M H₂SO₄ (QY_ref = 0.54) as the standard reference. Briefly, a series of dilute solutions of both the LLC sample and the quinine sulfate standard were prepared to ensure their absorbance values at the excitation wavelength (360 nm) were below 0.1, thereby minimizing inner filter effects. The fluorescence emission spectra of these solutions were then recorded using a fluorescence spectrophotometer (LS-45, PerkinElmer, Yokohama, Japan) with the same instrument parameters. The QY of LLC was calculated using the following equation:

where QY represents the fluorescence quantum yield of the sample under test; QY_ref represents the fluorescence quantum yield of the standard material; I represents the integrated fluorescence intensity of the sample under test; I_ref is the integrated fluorescence intensity of the standard substance; A is the maximum absorbance value of the test sample at the same excitation wavelength; A_ref is the maximum absorbance value of the standard substance’s diluted solution at the same excitation wavelength.

In vitro inverted coagulation experiment

Take a test tube, add 50 μL of sample solution (5 mg/mL LLC, 2 mg/mL LLC, 1 mg/mL LLC, 5 mg/mL LL, 2 mg/mL LL, 1 mg/mL LL) to the experimental group; add 50 μL of physiological saline to the negative control group; and add 50 μL of ultrapure water to the blank control group. All samples are pre-warmed at 37 °C for 5 min. Subsequently, add 400 μL of anticoagulated mouse blood pre-warmed at 37 °C to each tube, gently mix, and then add 0.1 M calcium chloride in a volume ratio of 10:1 to re-calcify the anticoagulated whole blood. Mix again and react at 37 °C for 5 min. After the reaction, invert the test tubes horizontally by 180° to observe the detachment and morphology of the blood clot.

Coagulation time determination

Add 100 μL of the sample solution to the bottom of each well in a 48-well plate, and then distribute 100 μL of citrated blood into each well of the 48-well plate. At predetermined time points, wash the plate with saline to terminate the coagulation reaction, and quickly aspirate and thoroughly wash until the solution becomes clear.

Hemodynamic measurement

Add 100 μL of the sample solution to the bottom of each well in a 48-well plate. Then, introduce 50 μL of citrated whole blood into the same plate and mix it with the sample. At each specified time point, add 2.9 mL of distilled water to each well and maintain it for 10 min to lyse the red blood cells. Measure the absorbance of the resulting supernatant at 540 nm using a microplate reader (Synergy H1, BioTek Instruments, Winooski, VT, USA).

Analysis of Platelet Count and Coagulation Function

Normal rats were randomly divided into 6 groups: saline group, low-dose LL (L-LL, 100 mg/kg) group, high-dose LL (H-LL, 300 mg/kg) group, low-dose LLC (L-LLC, 100 mg/kg) group, and high-dose LLC (H-LLC, 300 mg/kg) group. After 7 days of oral administration of LL and LLC, blood samples were collected from the abdominal aorta of anesthetized rats into tubes containing heparin anticoagulant. Platelet counts in whole blood were immediately analyzed using a fully-automated coagulation analyzer (YX-2000, Zhongyuan Huiji Biotechnology Co., Ltd., Chongqing, China). For the four coagulation parameters, blood samples were placed in tubes containing sodium citrate anticoagulant, then centrifuged at 3,000 rpm for 15 min to obtain platelet-poor plasma. Coagulation parameters, including prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB) concentration, were measured using a fully automated coagulation analyzer according to the manufacturer’s instructions.

Free radical scavenging experiment

The free radical scavenging experiment evaluates antioxidant activity through DPPH•, ABTS⁺•, •OH, and O₂⁻• scavenging assays. As a stable nitrogen-centered free radical, 0.5 mL of DPPH (0.4 mmol/L) reacts with 0.5 mL of LLC solutions at various concentrations (15.625, 31.25, 62.5, 125, 250, 500, 1000, and 2000 µg/mL) in the dark for 30 min, and the absorbance is measured at 517 nm.

In a dark environment, 0.8 mL of ABTS (7 mM) was mixed with 1 mL of potassium persulfate (K₂S₂O₈, 2.45 mM) and allowed to incubate overnight. The resulting ABTS⁺• was then incubated with LLC at the same concentration used in the DPPH• scavenging experiment. The absorbance value of ABTS⁺• was recorded at 738 nm.

In the •OH scavenging experiment, a Fenton reaction system was employed, in which 1 mL of methylene blue (1 mmol/L), 0.2 mL of FeSO₄ (100 mmol/L), 0.8 mL of 1% H₂O₂, and 0.05 mL of H₂SO₄ (0.5 mol/L) were mixed with different concentrations of LLC. The reaction was conducted at 37 °C for 15 min, and the absorbance was measured at 664 nm.

In the O₂⁻• radical scavenging experiment, 0.6 mL of 0.05 mol/L Tris–HCl buffer at pH 8.2 and 0.2 mL of the sample were added to each test tube. After incubating at 25 °C for 10 min, 40 μL of a pre-warmed 30 mmol/L pyrogallol solution, also at the same temperature, was added. The reaction was allowed to proceed for 4 min, after which 0.1 mL of concentrated hydrochloric acid was added to terminate the reaction. The absorbance was measured at a wavelength of 320 nm.

The absorbance of the sample after the reaction with the system is recorded as the sample tube (A1). To account for the absorbance effect caused by the background of the sample, a sample control tube (A2) is also set up. Additionally, a blank control tube (A0) that does not contain the sample is established. The free radical scavenging ability (%) is calculated according to Eq. (1).

In Vivo imaging of gastrointestinal accumulation

The gastrointestinal accumulation of LLC was evaluated in vivo using an IVIS^® Spectrum in vivo imaging system (PerkinElmer, Waltham, MA, USA). To accurately assess its gastrointestinal distribution and avoid interference from autofluorescence in the animals, we subjected the mice to a 24 h fasting period prior to dosing to ensure gastric emptying and reduce background signals. After intragastric administration of LLC, the mice were anesthetized with isoflurane and placed in the imaging chamber at predetermined time points (0.5, 1, 2, 4, 8, 12 and 24 h). The fluorescence signals within the gastrointestinal tract were captured and quantified using the system’s Living Image^® software. The signal intensity was represented by a color scale, with red and blue indicating the maximum and minimum radiance, respectively, to visualize the spatiotemporal distribution and accumulation of LLC.

Modeling and treatment of UC

SPF-grade C57BL/6 male mice, aged 6–8 weeks and weighing 22–25 g, were purchased from Spebio (Beijing) Biotechnology Co., Ltd. (License No.: SYXK(Yunnan)2024–0001). All animals were housed in the IVC observation room of the Dali University Laboratory Animal Center. All experimental studies were conducted in accordance with the requirements of the Dali University Animal Ethics Committee (Ethics Approval Number: 2025SL0034). A large number of male C57BL/6 mice were acclimated in standard housing conditions for one week prior to the experiment. They were housed individually in cages of eight mice each, with free access to food and water. Bedding was replaced and cages cleaned and disinfected regularly. Following the acclimation period, mice were stratified and randomized by body weight into a normal control group (Control, n = 6) and a model group. The experimental period lasted 14 days: Days 1–10: Modeling group mice freely drank a 2% (w/v) sodium dextran sulfate (DSS) solution (changed every 2 days) to establish an UC model. Starting on Day 10, drinking water was switched to pure water until the end of the experiment. Control group mice freely drank pure water throughout the entire period. On Day 8, all modeled mice underwent disease activity index (DAI) scoring. Based on inflammation severity, extremely mild and severe cases were excluded. The remaining successfully modeled mice were randomly assigned to 6 groups (n = 8): model group (Model), mesalazine group (200 mg/kg), Low-dose LL group (L-LL, 100 mg/kg), High-dose LL group (H-LL, 300 mg/kg), Low-dose LLC group (L-LLC, 100 mg/kg), and High-dose LLC group (H-LLC, 300 mg/kg). From Day 8 to 14, all mice received daily oral administration: control and model groups received saline (0.2 mL/20 g), while drug groups received their respective medications (dose: 0.1 mL/20 g). All behavioral observations and histological analyses were conducted under single-blind conditions, with scorers and analysts unaware of group assignments.

General observations of physical characteristics

During the experiment, the body weight of mice in each group was recorded daily. Changes in food and water intake, fur color, spontaneous activity, and overall mental state of the mice in each group were observed.

DAI scores

During the experimental period, the body weight of the mice was weighed and recorded daily. Fresh feces from the mice were collected and placed in a 24-well plate to observe and record the characteristics of the feces. Subsequently, a fecal occult blood test was conducted using an occult blood reagent kit, and the conditions of occult blood and visible blood were evaluated and recorded according to the instructions of the kit. The DAI scoring standard was referenced and improved based on the literature, and DAI scores were assigned to the mice in each group, as detailed in Table 1.

Table 1.

DAI scoring criteria (DAI = a + b + c)

Score	Weight loss (a)	Stool characteristics (b)	Fecal occult blood status (c)
0	≤ 1%	Normal	negative occult blood (-)
1	> 1% ~ ≤ 5%	Between normal and loose stool	positive occult blood (+)
2	> 5% ~ ≤ 10%	Loose stool	moderate occult blood (+ +)
3	> 10% ~ ≤ 15%	Between loose stool and diarrhea	significant occult blood (+ + +)
4	> 15%	Diarrhea	grossly visible blood in stool (+ + + +)

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Normal stool = formed feces; loose stool = unformed liquid feces; diarrhea = liquid feces adhering to the anus. Weight loss (%) = (Weight at a certain time after modeling—Weight before modeling)/Weight before modeling ×100%;

DAI = Weight loss score + Stool consistency score + Occult blood score (minimum score 0, maximum score 12)

Calculation of Organ Index

Mice were subjected to isoflurane-induced anesthesia, followed by rapid blood collection via ocular puncture and subsequent cervical dislocation for euthanasia. The spleen, thymus, and colon were excised, the surface blood was wiped off, and the organs were weighed to calculate the organ index.

Measurement of Macroscopic indicators related to the colon

The colon tissue is placed on a white paper with a ruler, and the length of the colon is measured and recorded. After rinsing the feces with physiological saline, the colon tissue is longitudinally dissected, and moisture is absorbed with filter paper. The tissue is then laid flat on the white paper for visual observation of the intestinal mucosal damage. The colon mucosa damage index (CMDI) is assessed according to the grading standards described in the literature [32], with the criteria presented in Table 2.

Table 2.

CMDI scoring criteria

Score	Colonic mucosa
0	No damage
1	Mild congestion and edema, with a smooth surface and no erosion or ulcers
2	Congestion and edema with rough mucosa, presenting a granular appearance with erosion or intestinal adhesion
3	Severe congestion and edema with a surface area of necrosis and ulcers < 1 cm², thickening of the intestinal wall or presence of necrosis and inflammatory polyps
4	Severe congestion and edema with mucosal necrosis and ulcer area > 1 cm² or total intestinal wall necrosis, leading to death from toxic megacolon

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H&E staining, PAS staining, and histopathological scoring

Colonic tissues were fixed in 10% formalin solution, embedded in paraffin, and sectioned for hematoxylin and eosin (H&E) staining as well as periodic acid-Schiff (PAS) staining. Histopathological studies were conducted under an optical microscope. Histopathological scoring was performed according to the standards established in reference [32], as detailed in Table 3. The sum of the scores for epithelial cell and inflammatory cell infiltration constitutes the the histological score (HS).

Table 3.

Histopathological scoring criteria

Score	Epithelial cells	Inflammatory cell infiltration
0	Normal morphology	No infiltration
1	Loss of goblet cells	Infiltration at the base of crypts
2	Extensive loss of goblet cells	Infiltration reaches the muscularis mucosa
3	Loss of crypt cells	Infiltration extends into the muscularis mucosa, accompanied by mucosal thickening and significant edema
4	Extensive loss of crypt cells	Infiltration reaches the submucosa

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Tail amputation experiment measured bleeding time and blood volume

On the last day of treatment, the mice were placed in a prone position and anesthetized with isoflurane. The tail was disinfected (wiped with 75% alcohol). The tail was then immersed in 40 °C water for 5 min, followed by a circular incision 1 cm from the tail tip to expose the caudal vertebra, which was then severed. Bleeding time was recorded as the duration (in seconds) from amputation to natural hemostasis. Blood volume was recorded as the mass (in mg) from amputation to natural hemostasis.

Detection of cytokine levels by ELISA

Remove colon tissue from the −80 °C freezer. After stepwise thawing at −20 °C and 4 °C, accurately weigh 50 mg of animal tissue. Under ice-water bath conditions, mechanically homogenize to prepare a 10% homogenate. volume (μl) = 1:9 ratio, add 9 times the volume of homogenization medium (0.9% saline). Centrifuge the homogenate at 4 °C and 3500 rpm for 10 min. Take the supernatant for BCA protein quantification kit (Beyotime, China). Simultaneously, prepare a standard curve based on the concentration and OD values of the standard samples. Subsequently, detect TNF-α, IL-1β, IL-6, IFN-γ, and IL-17 levels according to the ELISA kit instructions. Calculate sample concentrations using the standard curve equation and determine the corrected concentrations of each inflammatory factor.

Antioxidant measurement

After measuring the total protein concentration in the supernatant from item 2.16 using the BCA protein quantification kit, the expression levels of hydroxylphosphatidylcholine (HYP), myeloperoxidase (MPO), malondialdehyde (MDA), nitric oxide (NO), and reactive oxygen species (ROS) in the colon tissue homogenate were determined according to the operational procedures outlined in the kit manual. myeloperoxidase (MPO), malondialdehyde (MDA), nitric oxide (NO), and reactive oxygen species (ROS) in colon tissue homogenates.

CCD-841CoN cell culture and CCK-8 assay

Cells were cultured in DEME medium supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C in a humidified incubator with 5% CO₂. Passages were performed when cells reached 80–90% confluence.

Log-phase CCD-841CoN cells were seeded at 1 × 10⁴ cells/mL per well in a 96-well plate. Concentrations of mesalazine, LL, and LLC were added at 0, 6.25, 12.5, 25, 50, 100, and 200 µg/mL. with 5 replicate wells per concentration group. After 24 h of incubation in the cell culture incubator, CCK-8 was added. Following 1.5 h incubation, OD values were measured at 450 nm using a microplate reader (Synergy H1, BioTek Instruments, Winooski, VT, USA). Cell survival rates were calculated to determine the optimal intervention doses of mesalazine, LL, and LLC for this experiment.

Detection of p-NF-κB p65, NLRP3, caspase-1 and ASC mRNA expression in CCD-841CoN cells by RT-qPCR

We established an in vitro cellular inflammation model using UC cells and administered pharmacological interventions. Following published protocols [33, 34], CCD-841CoN cells cultured to logarithmic growth phase were seeded into 12-well plates using trypsin digestion solution at 1 mL per well. After 24 h of attachment at 37 °C in a 5% CO₂ incubator, the medium was removed. Except for the control group, all other groups were treated with final concentrations of 100 µg/mL LPS and 100 ng/mL TNF-α, followed by 24 h of incubation. After discarding the medium, the control and model groups were replenished with an equal volume of fresh medium. The drug intervention groups were treated with equal volumes of suspension containing final concentrations of 12.5 µg/mL mesalazine and 6.25, 12.5 or 25 µg/mL LL/LLC, respectively. Cultures were continued in a 37 °C, 5% CO₂ incubator for 48 h.

After 48 h of co-culture, cells were harvested. Total RNA was extracted using the Trizol method. Using PCR reverse transcription reagents provided by Yugong Biotech, total RNA was reverse transcribed into cDNA via a gene amplifier. Subsequently, real-time fluorescent quantitative PCR was performed to amplify target genes. GAPDH served as the internal control. Relative expression levels of p-NF-κB p65, NLRP3, Caspase-1, and ASC were calculated. Primer sequences are listed in Table S1.

Western blot detection of NLRP3/caspase-1/GSDMD protein expression in colon tissue

Approximately 50 mg of tissue was weighed and homogenized thoroughly on ice in RIPA lysis buffer supplemented with protease inhibitors to extract total proteins. The homogenate was centrifuged at 12,000 × g and 4 °C for 15 min, and the supernatant was collected subsequently. The total protein concentration in the supernatant was quantified using the BCA assay. Equal volumes of protein samples were mixed with 5 × SDS loading buffer, denatured by boiling, and then subjected to SDS-PAGE for protein separation. Separated proteins were transferred onto PVDF membranes. The membranes were blocked with TBST containing 5% skimmed milk powder at room temperature for 2 h. Following blocking, the membranes were incubated overnight at 4 °C with primary antibodies, including rabbit anti-mouse antibodies against nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3), cysteinyl aspartate specific proteinase-1 (Caspase-1), gasdermin D (GSDMD), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); the dilution ratios of primary antibodies were performed according to the manufacturers’ instructions. On the subsequent day, the membranes were washed three times with TBST, followed by incubation with corresponding HRP-conjugated secondary antibodies at room temperature for 2 h. After thorough washing with TBST again, ECL detection reagent was added to the membranes, and the protein bands were visualized using a chemiluminescence imaging system (GelView6000ProⅡ). Finally, the gray values of target protein bands were analyzed using ImageJ software, and the relative expression level of each target protein was calculated as the ratio of the gray value of the target protein to that of the internal reference protein GAPDH.

Immunofluorescence Detection of NLRP3, caspase-1, GSDMD and ASC protein expression in mouse colon tissue

Colon tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned into 5 μm thick slices. Following dewaxing, rehydration, antigen retrieval, and washing, sections were blocked with goat serum at room temperature for 30 min. Primary antibodies were applied sequentially: NLRP3 (GB114320, 1:400, servicebio), GSDMD (GB154198, 1:500, servicebio), Caspase-1 (GB15383, 1:2000, servicebio), and apoptosis‑associated speck‑like protein containing a CARD (ASC, GB153966, 1:500, ServiceBio) at room temperature overnight at 4°C. After washing, add HRP-labeled secondary antibody (GB23404, 1:500, ServiceBio) and incubate at room temperature in the dark for 1 h. Following washing with fluorescent signal amplification buffer, perform DAPI staining. Incubate at room temperature in the dark for 10 min, then wash. Allow sections to air-dry slightly, then mount with anti-fade mounting medium. Capture all fluorescence images using a Pannoramic MIDI scanner (3D HISTECH, Budapest, Hungary). Perform semi-quantitative analysis of fluorescence intensity using ImageJ software.

Detection of CD3⁺/CD4⁺/CD8⁺ T lymphocyte levels and cytokine levels of IL-17 and treg in the spleen and lymph nodes by flow cytometry

The spleen and lymph node tissues were ground into single-cell suspensions. After erythrocyte lysis, flow cytometry (FACSCanto II, BD Biosciences, Franklin Lakes, NJ, USA) was used for detection. For CD3⁺/CD4⁺/CD8⁺ T lymphocytes, corresponding fluorescently labeled antibodies were used for labeling, and the proportions of each subset were analyzed by flow cytometry. Meanwhile, fluorescently labeled antibodies specific to IL-17 and Treg were used to detect the expression levels of these two cytokines in the spleen and lymph nodes.

Immunofluorescence staining of Claudin-1, occludin and ZO-1

The immunofluorescence staining of tight junction proteins in the mouse colon was performed. Paraffin-embedded colon sections were incubated overnight at 4 °C with primary antibodies against Claudin-1 (GB15032, 1:4000, Servicebio), Occludin (GB111401, 1:3000, Servicebio), and ZO-1 (GB151981, 1:3000, Servicebio). Following this, the sections were incubated at 37 °C for 50 min with HRP-conjugated goat anti-mouse IgG (GB23301, 1:500, Servicebio) and HRP-conjugated goat anti-rabbit IgG (GB23303, 1:500, Servicebio). Subsequently, the corresponding TSA was added, followed by microwave treatment and serum blocking, repeated twice. The nuclei were counterstained with DAPI, and then a fluorescence quenching agent was applied and washed for 10 min. Finally, the sections were mounted with an anti-fluorescence quenching mounting medium (G1401) and all fluorescence images were captured using a panoramic tissue section scanner (Pannoramic MIDI, 3DHISTECH Ltd., Budapest, Hungary).

Analysis of gut microbiota

Following the manufacturer’s instructions, microbial DNA was extracted from mouse fecal samples in each treatment group using the VAMNE Stool/Soil DNA Extraction Kit-BOX 2 (Vazyme Biotech Co., Ltd.). The DNA concentration was accurately quantified using Qubit, and the integrity of DNA fragments was assessed using the Qsep400 high-throughput biofragment analyzer. Qualified DNA samples were fragmented to approximately 300 bp using enzyme digestion, followed by end repair, A-tailing, adapter ligation, purification, and PCR amplification to complete the library preparation. Once the library was constructed, the insert size was verified using Qubit 4.0 and Qsep400. After confirming the quality of the library, different libraries were pooled according to the quality concentration and the required data volume for downstream analysis, followed by single-strand circularization and preparation of DNB nanoparticles for PE150 sequencing. The raw sequencing data was processed to obtain clean data, further revealing low-abundance species information within the samples and generating a non-redundant Unigenes set along with abundance information of Unigenes across various samples. The Unigenes were compared with the MicroNR database to obtain species classification information at various taxonomic levels. Based on the statistical results of species annotation at the species level, analyses such as Alpha and Beta diversity were conducted to assess the diversity and structural differences of gut microbiota among different treatment groups of mice.

Statistical Methods

Data were analyzed using SPSS 25.0, and results are presented as mean ± SD (standard deviations). One-way analysis of variance (ANOVA) was used for comparison among groups. If the difference was significant, back testing corrected by Tukey method was further used for pairwise comparison. p < 0.05 is considered statistically significant.

Results

Discovery and characterization of CDs in LLC

Carbonization temperature and time are two critical parameters in the carbonization process. To determine the optimal conditions for LLC, a systematic parameter screening was conducted by varying the carbonization temperature (350, 450, and 550 ℃) and time (0.5, 1 and 2 h). The resulting LLC samples were evaluated based on CY, elemental composition (XPS), surface functional groups (FTIR), QY, colloidal stability (Zeta potential), and in vitro antioxidant capacity (DPPH and ABTS⁺ assays). As shown in Fig. S1-S8, with increasing temperature and time, the carbon content of LLC rises while oxygen and nitrogen contents decrease; CY gradually declines, while QY increases. Samples prepared at 450 ℃ for 1 h exhibited a higher carbonization yield (38.1%) compared to those processed at 550 ℃, achieved a higher quantum yield (2.3%) than those processed at 350 ℃, and moderately retained oxygen- and nitrogen-containing functional groups crucial for dispersion and biological activity. This likely explains its superior radical scavenging activity compared to the high-temperature treatment group. Additionally, this condition demonstrated higher energy efficiency. Therefore, treatment at 450 °C for 1 h was determined as the optimal preparation condition of LLC.

As shown in Fig. 1A, LLC was prepared via high-temperature pyrolysis. Under sunlight, LLC exhibits a yellow–brown color and displays a distinct tyndall effect. Interestingly, when illuminated with 365 nm excitation light, the solution emits a blue-green photoluminescence (Fig. 1B). The HRTEM images clearly demonstrate their excellent dispersion and quasi-spherical morphology (Fig. 1C). it was found to contain a large number of spherical nanoparticles uniformly distributed in the range of 0.5–3 nm. Statistical analysis of over 100 particles revealed an average particle size of 1.72 ± 0.3 nm, with a lattice spacing of 0.26 nm (Fig. 1D), consistent with carbon dot structures. One study found that the carbon dots derived from Fuligo Plantae have a particle size distribution ranging from 1.4 to 3.2 nm and exhibit good permeability across the gastric mucosal barrier after oral administration, effectively improving tissue damage and inflammation [35]. This suggests that the small-sized carbon dots rich in LLC may confer excellent biological barrier penetration capabilities, potentially resulting in remarkable therapeutic effects. Figure 1E shows the FE-SEM image of LLC and the corresponding element distribution map. The signal distribution of element C is the most extensive and the intensity is the highest. At the same time, O and N elements also show a uniform distribution state, and highly overlap with the distribution area of carbon elements. The UV–visible spectrum of the LLC solution exhibits a weak absorption band around 280 nm (Fig. 1F), which is attributed to the π-π* electronic transitions of the conjugated C = C bonds and aromatic sp² domains on the carbon core [36]. FTIR (Fig. 1G) shows that LLC has obvious absorption peaks at wave numbers of 3400 cm⁻¹, 2920 cm⁻¹, 1710 cm⁻¹ and 1650 cm⁻¹, which are attributed to the stretching vibration of O–H/N–H, C-H, C = O and C = C bonds respectively. In the FL spectrum (Fig. 1H), the maximum excitation wavelength and maximum emission wavelength are 390 nm and 501 nm, respectively.The influence of different excitation wavelengths on the emission wavelength of carbon dots is discussed (Fig. 1I). As the excitation wavelength varies within the range of 250 to 370 nm, a redshift in the emission wavelength is observed. Figure 1J shows the fluorescence lifetime decay curve of LLC, which follows a double exponential decay model. The average fluorescence lifetime is 5.98 ns. As shown in Fig. 1K, the peaks of ¹H in aliphatic hydrocarbons appear in the range of 1.0–1.8 ppm, while the peak at approximately 8.5 ppm can be attributed to ¹H in the aromatic ring. The XRD spectrum of LLC shows (Fig. 1L) a broad reflection peak centered at 20.6°, which corresponds to the (002) diffraction plane attributed to disordered carbon atoms in graphite. As shown in Fig. 1M, the Raman spectrum of LLC exhibits two peaks at 1590 and 1350 cm⁻¹, corresponding to the G band and D band of graphene, respectively. The intensity ratio of the D band to the G band is approximately 1.0, indicating that a substantial portion of the carbon dots in LLC contains defects. Additionally, XPS was employed to investigate the final composition of the carbon dot surface (Fig. 1N), revealing three main peaks corresponding to C (81.49%), O (16.08%), and N (2.43%). The O 1 s spectrum (Fig. 1O) confirms that the peaks correspond to C-O (531.8 eV) and C = O (533.1 eV). The C 1 s spectrum (Fig. 1P) confirms the presence of C–C/C = C (284.4 eV), C-O/C–N (286.2 eV), and C = O (288.6 eV). The N 1 s spectrum (Fig. 1Q) also shows the presence of pyrrolic nitrogen (399.6 eV) and graphitic nitrogen (400.6 eV) within the carbon dots.

Hemostatic effect of LLC

The procoagulant activity of LLC was systematically evaluated through in vitro and in vivo experiments. In the in vitro assay (Fig. 2A), LLC exhibited a dose-dependent procoagulant effect, achieving complete blood coagulation at a concentration of 5 mg/mL, whereas LL at the same concentration resulted only in partial clotting (Fig. 2B). Further validation using a 48-well plate coagulation time assay (Fig. 2C) confirmed that LLC formed a uniform, dark red, and stable blood clot in a shorter time compared to LL (Fig. 2D). Additionally, coagulation dynamics experiments demonstrated that the clots induced by LLC showed a lower degree of dissolution in distilled water (Fig. 2E), indicating that LLC not only accelerates the coagulation process but also enhances clot stability.

To evaluate systemic effects in vivo, rats received oral LLC for seven days, after which blood was collected for analysis of platelet counts and plasma coagulation parameters (Fig. 2F). High-dose LLC (H-LLC) significantly increased peripheral platelet levels compared with both control and LL groups (Fig. 2G), suggesting enhanced platelet production and greater availability for primary hemostasis. Further analysis of coagulation function (Fig. 2H) revealed that LLC intervention markedly altered key coagulation parameters. Specifically, compared with the control, the LLC‑treated groups (particularly H‑LLC) showed a significant increase in FIB concentration and a pronounced shortening of both APTT and TT, with effects that were significantly stronger than those of the low‑dose LL group. In contrast, no significant change was observed in PT. The elevated FIB level indicates enhanced capacity for fibrin‑mesh formation, which contributes to a more stable clot [37]. The shortening of APTT and TT, which reflect the activity of the intrinsic and common coagulation pathways, respectively, suggests that LLC accelerates key steps of the coagulation cascade, particularly by activating the intrinsic pathway and promoting thrombin generation, thereby improving overall coagulation efficiency [38].

The combined results demonstrate that oral administration of LLC effectively increases platelet count in mice and promotes a pro‑coagulant trend, likely by elevating fibrinogen levels and accelerating the intrinsic coagulation pathway. These findings align with previous reports on the hemostatic potential of carbonized herbal materials [39, 40].

Antioxidant activity and gastrointestinal retention capacity of LLC

Given the strong pro-coagulant activity of LLC, we next examined its potential as a potent reactive oxygen species (ROS) scavenger, as broad-spectrum antioxidant capacity is crucial for alleviating UC. We evaluated LLC’s ability to scavenge DPPH•, ABTS⁺•, •OH, and O₂⁻• radicals (Fig. 3A). As shown in Fig. 3B, radical scavenging increased with LLC concentration. At 2000 µg/mL, LLC achieved scavenging rates of 107.6% for DPPH•, 112.6% for ABTS⁺•, 99.8% for •OH, and 84.6% for O₂⁻•, confirming its robust antioxidant activity.

To assess its suitability as an oral therapeutic, we further investigated LLC’s stability in physiological environments, its antioxidant performance under these conditions, and its gastrointestinal retention in vivo. LLC remained well dispersed without aggregation in phosphate-buffered saline (PBS), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) (Fig. 3C). Zeta potential measurements showed a consistently high negative surface charge across all three media (Fig. 3D), indicating strong electrostatic stabilization and suggesting that LLC can maintain colloidal integrity throughout the gastrointestinal tract. Antioxidant assays in these media revealed environment-dependent activity. In the DPPH assay (Fig. 3E), LLC showed enhanced scavenging in SGF and SIF compared with PBS, suggesting the gastrointestinal milieu may boost its efficacy against DPPH radicals. In contrast, ABTS⁺ scavenging (Fig. 3F) was slightly reduced in SGF but improved in SIF, highlighting differential modulation by gastric versus intestinal conditions. In different physiological simulants, LLC exhibits varying scavenging activities against DPPH and ABTS⁺ radicals, primarily attributed to the sensitivity of its antioxidant mechanisms to environmental pH. DPPH scavenging relies mainly on hydrogen atom transfer, with enhanced activity in both acidic and weakly alkaline environments. This may correlate with changes in the reactivity of surface groups on LLC and improved dispersion at different pH levels. Conversely, ABTS⁺ scavenging primarily occurs via electron transfer pathways. In strongly acidic conditions, its electron-donating capacity is inhibited, leading to a slight decrease in activity. Conversely, in the weakly alkaline simulated intestinal environment, LLC’s electron transfer ability is significantly enhanced, resulting in increased activity. This finding suggests that LLC maintains and exerts more comprehensive antioxidant efficacy in its primary site of action after oral administration—the intestinal environment.

In vivo imaging revealed (Fig. 3G–H) that LLC fluorescence signals reached peak intensity in the intestines within 1 h post-oral administration. Subsequently, signals migrated gradually toward the intestine with gastrointestinal motility, showing marked enrichment in the small intestine and colon regions. Signal intensity gradually declined after peaking, with most signals metabolized and cleared by 12 h. Notably, however, distinct fluorescent residues remained detectable in the colonic region 24 h post-administration, indicating sustained retention capacity of LLC at this site.

LLC alleviates UC

An acute colitis model was successfully established via DSS induction (Fig. 4A). Model mice exhibited typical disease phenotypes, including weight loss, hematochezia, and diarrhea, accompanied by a significant increase in the DAI score by day 7 (Fig. 4B -C, Fig. S9). Treatment with LL and LLC effectively mitigated body weight loss and reduced DAI scores, with H-LLC showing the most pronounced restorative effect by the end of the study (day 14).

DSS-induced colitis led to thymic atrophy and splenomegaly, as indicated by organ indices (Fig. 4D-E). Both LL and LLC treatments reversed these changes, and notably, the spleen index in the H-LLC group was significantly lower than in other treatment groups, suggesting a potent modulatory effect on systemic immune imbalance.

Macroscopic evaluation of colon morphology revealed severe damage in the Model group, including significant colon shortening, mucosal congestion, edema, and ulceration, reflected by a decreased colon index and a markedly increased CMDI score (Fig. 4F-K). All treatments ameliorated these pathological alterations. Particularly, H-LLC treatment resulted in the greatest restoration of colon length, the most improved mucosal appearance (minimal congestion and smooth surface), and the lowest CMDI score, demonstrating superior efficacy in promoting mucosal healing.

LLC alleviates pathological damage in mice with UC

To further evaluate the therapeutic effect of LLC, colon tissues were analyzed by H&E and PAS staining. H&E staining (Fig. 5A) revealed that DSS induction caused severe colonic injury, characterized by crypt distortion and atrophy, extensive inflammatory cell infiltration into the submucosa, mucosal edema, and near-complete loss of goblet cells. These features reflect a profound disruption of the epithelial barrier, a hallmark of UC pathogenesis. Accordingly, the HS in the Model group was significantly elevated compared with the Control group. All treatment regimens ameliorated these pathological changes and reduced the HS. Notably, the H-LLC group exhibited the most pronounced recovery, with near-normal crypt architecture, substantial restoration of goblet cell numbers, and minimal inflammatory infiltration.

PAS staining (Fig. 5B) further confirmed impaired mucus production in the Model group, as evidenced by marked depletion of mucin-containing goblet cells. In contrast, LLC treatment, particularly at the high dose, effectively restored goblet cell density and mucin secretion. This indicates that LLC not only suppresses inflammation but also actively supports the regeneration of the protective mucus layer, which is essential for maintaining intestinal homeostasis and preventing bacterial translocation.

These findings are consistent with current understanding that effective UC therapies should go beyond anti-inflammatory effects to promote mucosal healing. The superior performance of H-LLC over LL suggests a dose-dependent benefit, and its dual action on both inflammation and epithelial repair may offer advantages over conventional agents such as mesalazine, which primarily target inflammatory mediators without directly enhancing barrier restoration. Moreover, histopathological evaluation of major organs including the heart, liver, spleen, lungs, and kidneys showed no evidence of toxicity, such as cellular degeneration, necrosis, or inflammatory infiltration, in any of the treatment groups (Fig. S10).

Collectively, these results demonstrate that LLC exerts significant therapeutic effects in DSS-induced colitis by mitigating tissue damage, restoring goblet cell function, and reinforcing mucosal barrier integrity, all while exhibiting a favorable safety profile.

Hemostatic properties of LLC

During UC progression, intestinal bleeding and diarrhea exacerbate inflammation and increase treatment complexity [41]. To evaluate the potential of LLC in controlling bleeding in vivo, a DSS-induced UC model was established, and diarrhea/bloody stool scores were recorded (Fig. 6A). After 7 days of DSS administration, all model mice exhibited diarrhea and fecal occult blood. Following treatment, these symptoms were alleviated, with the L-LLC and H-LLC groups showing the most pronounced improvement by day 14 (Fig. 6B-C). UC is often accompanied by impaired platelet function and coagulation. Tail‑transection assays revealed that the Model group had significantly prolonged bleeding time and increased blood loss compared to the Control (Fig. 6D-E). Treatment with LL and LLC significantly shortened the bleeding time and reduced blood loss, with H-LLC showing efficacy comparable to the positive control. Notably, the hemostatic effect of LLC was markedly stronger than that of LL.

Fig. 6 — Hemostatic effects of oral LLC on mice with UC. (A) Representative photographs of wet tails and hematochezia on days 7 and 14. (B) Diarrhea index on day 14 and (C) hematochezia index. (D) Blood loss in the tail amputation model and (E) hemostatic time. (n = 6). (^#p < 0.05, ^## p < 0.01, ^###p < 0.001 vs. Control; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Model; ^ap < 0.05, ^aap < 0.01, ^aaap < 0.001 vs. H-LLC)

This enhanced activity may be attributed to the carbonaceous nanomaterials formed during pyrolysis, consistent with previous reports indicating carbon dots as key hemostatic components in carbonized traditional Chinese medicine [42]. Moreover, these in vivo hemostatic effects align with our earlier systemic coagulation analysis, which demonstrated that oral administration of LLC increases platelet counts and enhances key coagulation parameters, including elevated fibrinogen levels and accelerated intrinsic pathway activity. Therefore, the potent hemostatic properties exhibited by LLC in vitro and in vivo likely originate from its carbon dot material.

In Vivo anti-inflammatory and antioxidant effects of LLC

To further evaluate whether LLC administration can downregulate the inflammatory response in UC lesions, several key UC-related cytokines were measured. As shown in Fig. 7A, myeloid dendritic cells are activated by key cytokines produced by immune cells, presenting antigens and releasing substances, including interleukin-12 (IL-12) and interleukin-23 (IL-23), which have been confirmed to trigger the formation of Th17 and Th1 helper T cells [43]. Subsequently, these T cells release factors such as interleukin-17A, IFN-γ, and interleukin-22 (IL-22), stimulating colonic epithelial cells to produce pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6).

The inflammatory cytokine profile in colon tissue was assessed by ELISA (Fig. 7B-F). Compared to the Control, the Model group showed a significant upregulation of all tested pro-inflammatory mediators (TNF-α, IL-6, IL-1β, IFN-γ, IL-17). Treatment with LLC effectively suppressed this overexpression. A key finding was the superior performance of H-LLC, which achieved a more pronounced reduction in key cytokines such as TNF-α, IL-6, and IFN-γ than equivalent or lower doses of the LL. This indicates that LLC, through its potent suppression of the inflammatory cascade, offers a distinct advantage in mitigating intestinal inflammation and restoring immune balance. The oxidative and fibrotic state of colon tissue was assessed via key biomarkers (Fig. 7G-J). The Model group showed a substantial upregulation of oxidative stress markers (ROS, MDA, NO) and the fibrosis indicator HYP, confirming severe oxidative damage and aberrant collagen deposition. Pharmacological intervention reversed these alterations. Importantly, H-LLC outperformed both LL and L-LLC, showing superior efficacy in suppressing ROS/MDA/NO and normalizing HYP levels. These results demonstrate that H-LLC can more effectively counteract oxidative stress and inhibit the associated fibrotic progression in UC.

Effects of LLC on NLRP3/caspase-1/GSDMD-mediated pyroptosis pathway

In recent years, the role of pyroptosis‑mediated inflammatory responses in the pathological process of UC has attracted considerable attention. Pyroptosis is a caspase‑dependent, inflammatory form of programmed cell death [44]. Characterized by NLRP3 inflammasome assembly, caspase‑1 activation, and cleavage of GSDMD, pyroptosis is highly expressed in the colon tissues of UC patients, confirming its close association with the pathogenesis of UC [45]. Studies have shown that inhibiting pyroptosis can alleviate damage in experimental colitis [46]. Although traditional charcoal‑processed drugs have been used in UC, no studies have yet reported whether LLC exerts therapeutic effects on UC by suppressing pyroptosis. Therefore, on the basis of confirming the efficacy of LLC in treating UC, this study further investigates the effect of LLC on pyroptosis mediated by the NLRP3/caspase‑1 pathway in an LPS and TNF‑α‑induced in vitro inflammatory model (CCD‑841CoN cells) and in a DSS‑induced UC mouse model, aiming to preliminarily elucidate its underlying mechanism.

We first assessed the cytotoxicity of mesalazine, LL, and LLC on CCD-841CoN cells using the CCK8 assay (Fig. 8A). Results shown in Fig. 8B indicate that at concentrations ranging from 6.25 to 200 μg/mL, mesalazine, LL, and LLC exhibited no significant cytotoxic effects compared to the control group at the concentrations tested. Furthermore, the LL group exhibited a cell proliferation-promoting effect. Subsequently, mesalazine at 12.5 μg/mL and LL/LLC at three concentrations (25, 12.5, and 6.25 μg/mL) were selected for intervention in CCD-841CoN cells. Subsequently, an in vitro UC model was established by stimulating CCD-841CoN cells with LPS and TNF-α (Fig. 8C). After 48 h of co-culture with different drug concentrations, mRNA expression levels of key pyroptosis pathway proteins in CCD-841CoN cells were detected via qRT-PCR. The results of qRT-PCR (Fig. 8D) showed that compared to the Control group, the Model group exhibited significantly elevated mRNA levels of p-NF-κB p65, NLRP3, Caspase-1, and ASC, indicating marked activation of the inflammasome pathway. After 48 h of treatment, all drug groups demonstrated significantly reduced mRNA levels of p-NF-κB p65, NLRP3, Caspase-1, and ASC mRNA levels were significantly reduced in all treatment groups after 48 h of treatment. LLC at 25 μg/mL demonstrated superior efficacy in reducing p-NF-κB p65, Caspase-1, and ASC mRNA levels compared to LL at 25 μg/mL. Results indicate that LLC significantly inhibits NF-κB pathway activation (downregulating p-NF-κB p65 mRNA) and upstreamly downregulates gene expression of pyroptosis core components NLRP3, ASC, and Caspase-1.

Subsequently, we examined the expression levels of key pyroptosis pathway proteins in colonic tissue via Western Blot and immunofluorescence. As shown in Fig. 9A-B, compared to the Control group, NLRP3, Cleaved Caspase-1, and GSDMD-NT protein expression levels were significantly elevated in the Model group colonic tissue, indicating successful activation of the NLRP3 inflammasome-mediated pyroptosis pathway by DSS modeling. Following pharmacological intervention, both the positive control drug Mesalazine and LLC treatment effectively reversed this trend, significantly downregulating the expression of the aforementioned key proteins. The H-LLC group exhibited the strongest inhibitory effect, demonstrating a certain degree of dose dependency, while the effects of the L-LL and H-LL groups were relatively limited. Immunofluorescence staining (Fig. 9C-D) further confirmed this regulatory effect in situ. Fluorescence signal intensity for NLRP3, ASC, Caspase-1, and GSDMD was significantly enhanced in the Model group. Following drug treatment, fluorescence signals for these proteins were markedly suppressed, with LLC exhibiting superior regulatory effects compared to LL and demonstrating a dose-dependent response.

Fig. 9 — Effects of LLC on NLRP3 inflammasome pathway and pyroptosis-related protein expression in DSS-induced UC mouse colon tissue. (A) Western blot analysis of NLRP3, GSDMD, Caspase-1, and GAPDH (internal control) in colon tissue from each group (n = 3). (B) Quantitative statistical analysis of target protein expression levels in the figure. (C) Representative images of immunofluorescence staining for NLRP3, ASC, Caspase-1, and GSDMD in colon tissue sections from each group (n = 6, magnification at 200 ×). (D) Semi-quantitative analysis of fluorescence staining intensity in the figure. (^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Control; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Model; ^a p < 0.05, ^aap < 0.01, ^aaap < 0.001 vs. H-LLC)

In summary, LLC may suppress pyroptosis in colonic epithelial cells by inhibiting excessive activation of the NLRP3/Caspase-1/GSDMD signaling pathway, potentially representing a key mechanism underlying its anti-inflammatory effects in UC mice.

Regulation of immune homeostasis by LLC in mice with UC

The spleen, as a key peripheral immune organ, exhibited significant alterations in T lymphocyte subsets during UC progression. Compared to the Control group, DSS-induced mice showed a marked decrease in splenic CD3⁺and CD4⁺T cells, accompanied by a significant increase in CD8⁺T cells (Fig. 10A-C). This imbalance indicates systemic immune dysregulation. Both the positive control (Mesalazine) and LLC treatments reversed these changes. Notably, LLC dose-dependently restored the levels of CD4⁺T cells and the CD4⁺/CD8⁺ ratio while reducing CD8⁺T cell levels, demonstrating a superior corrective effect compared to the LL.

Fig. 10 — Immune homeostasis regulation in UC mice after oral LLC administration. (A) Flow cytometry of CD3⁺ cells in the spleen of mice. (B) Flow cytometry of CD4⁺CD8⁺ cells in the spleen of mice. (C) The effect on CD3⁺, CD4⁺, and CD8 + levels and the CD4⁺/CD8⁺ ratio in the spleen of mice. (D) Flow cytometry of Th17 cells in the spleen of mice. (E) Flow cytometry of Treg cells in the spleen of mice. (F) Flow cytometry of Th17 cells in the lymph of mice. (G) Flow cytometry of Treg cells in the lymph of mice. (H) The effect of LLC on the levels of Th17 and Treg cells and the Th17/Treg ratio in the spleen and lymph of mice. (n = 3). (^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Control; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Model; ^ap < 0.05, ^aap < 0.01, ^aaap < 0.001 vs. H-LLC)

Furthermore, DSS modeling disrupted the critical balance between pro-inflammatory Th17 cells and anti-inflammatory regulatory T cells (Tregs) in the spleen and lymph nodes. LLC administration potently counteracted this shift, significantly downregulating Th17 cells and upregulating Tregs, thereby effectively restoring the Treg/Th17 balance to a level comparable to that achieved by Mesalazine (Fig. 10D-H).

The Th17/Treg balance serves as a central hub for maintaining intestinal immune homeostasis, and its disruption directly drives mucosal inflammation [47]. Therefore, the restoration of the Th17/Treg balance by LLC not only reflects its systemic immunomodulatory capacity but also directly points to one mechanism underlying its alleviation of intestinal inflammation: namely, the re-establishment of immune tolerance and the suppression of excessive inflammatory responses. Such systemic regulation of adaptive immunity has not yet been reported in existing studies on herbal carbon dots for the treatment of UC.

LLC Restores intestinal barrier function

Claudin-1, Occludin and ZO-1 are core components of tight junctions that maintain the integrity of the intestinal epithelial barrier [48]. Under inflammatory conditions, their expression is frequently reduced, leading to increased paracellular permeability and facilitating the translocation of luminal antigens, which can further amplify mucosal inflammation. Immunofluorescence analysis showed that administration of 2% DSS markedly decreased the expression and disrupted the continuous membrane localization of Claudin-1, Occludin, and ZO-1 in colonic tissue (Fig. 11A-B), consistent with barrier dysfunction in experimental colitis.

Fig. 11 — LLC restores intestinal barrier function. (A) Fluorescent images of Occludin-1, Claudin-1, and ZO-1 in colonic tissue (magnification at 200 ×). (B) Relative expression levels of Occludin-1, Claudin-1, and ZO-1 in colonic tissue. (n = 6). (^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Control; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Model; ^ap < 0.05, ^aap < 0.01, ^aaap < 0.001 vs. H-LLC)

Treatment with LL or LLC partially restored the expression and distribution of these tight junction proteins. The H-LLC group exhibited the most evident recovery, with protein localization patterns closely resembling those observed in the Control group. This effect may be linked to the reduction in pro-inflammatory cytokines such as TNF-α and IFN-γ (Fig. 7), both of which have been shown to suppress tight junction protein expression through NF-κB–dependent signaling [49]. In addition, the attenuation of oxidative stress markers following LLC treatment may also support barrier integrity, as reactive oxygen species can disrupt tight junction assembly via post-translational modifications of junctional proteins [50].

The concurrent improvement in inflammation, oxidative stress, and epithelial barrier structure suggests that LLC acts on multiple interconnected aspects of UC pathophysiology. Restoration of tight junction proteins likely contributes to decreased intestinal permeability, thereby limiting antigenic exposure and downstream immune activation. These findings indicate that enhancing epithelial barrier function is an important dimension of LLC’s therapeutic activity in experimental colitis.

Regulation of gut Microbiota by LLC

UC is associated with gut microbial dysbiosis. In DSS-induced colitis, excessive epithelial necrosis disrupts the intestinal barrier, increasing permeability and allowing bacterial translocation into the lamina propria and bloodstream. This dysbiosis further impairs barrier function, creating a self-perpetuating cycle that exacerbates inflammation[51]. To evaluate whether H-LLC modulates the gut microbiota, we analyzed fecal samples from control, DSS, H-LL, and H-LLC treated mice.

The α-diversity, measured by Shannon and Simpson indices, was significantly reduced in the Model group but restored in both treatment groups, with H-LLC showing greater improvement (Fig. 12A and B). The β-diversity analysis using Bray–Curtis-based principal coordinate analysis (PCoA) revealed distinct microbial clustering among all four groups. PERMANOVA confirmed significant differences between Control and Model (R² = 0.456, p = 0.009), Model and H-LLC (R² = 0.543, p = 0.003), and H-LL and H-LLC (R² = 0.296, p = 0.013) (Fig. 12C).

At the phylum level, DSS decreased Bacillota and increased Bacteroidota, Deferribacterota, and Pseudomonadota (Fig. 12D, Fig. S11). H-LLC reversed these changes by suppressing Deferribacterota and Pseudomonadota, restoring Actinobacteriota, and significantly enriching Verrucomicrobiota—a beneficial taxon linked to mucosal health [52].

At the genus and species levels, DSS elevated Prevotella, Phocaeicola, and Mucispirillum, along with Phocaeicola vulgatus and Mucispirillum schaedleri, which are known to impair barrier integrity [53–55]. In contrast, H-LLC increased Bacteroides, Barnesiella, and Alistipes, and specifically enriched beneficial species such as Bacteroides acidifaciens [56], Barnesiella_sp_WM24 [57], and Parabacteroides distasonis [58]. These effects more pronounced than those of H-LL (Fig. 12E–H, Fig. S12–S13). H-LLC also restored members of Spirochaetaceae and Muribaculaceae, further supporting a healthier microbial profile [59].

LEfSe analysis identified five species significantly enriched by H-LLC compared with the Model group: Muribaculaceae_bacterium_Isolate_004_NCI, Barnesiella_sp_WM24, Muribaculaceae_bacterium_Isolate_013_NCI, Muribaculaceae_bacterium, and Bacteroides_acidifaciens—all associated with anti-inflammatory and barrier-repair functions (Fig. 12I, Fig. S14). When compared directly to H-LL, H-LLC uniquely promoted B. acidifaciens, Barnesiella_sp_WM24, and P. distasonis, whereas H-LL retained higher levels of potentially harmful taxa such as Clostridium_sp_MD294 (Fig. 12J, Fig. S15).

In summary, H-LLC significantly increased microbial diversity, rebalanced community composition, and shifted the gut ecosystem toward a symbiotic state, which was characterized by an increase in beneficial bacteria and a decrease in pathogenic populations. These microbial alterations were associated with improved barrier function, suggesting a potential link that requires further validation through fecal microbiota transplantation or germ-free animal experiments.

Discussion

UC is typically characterized by symptoms of colonic inflammation and bloody diarrhea. Considering the pathological features of UC, it is primarily defined by persistent inflammation of the rectal and colonic mucosa, often accompanied by oxidative stress. Clinically, this manifests as bloody diarrhea and mucosal ulcers [1]. Based on the close alignment between the hemostatic, anti-inflammatory, antioxidant, and intestinal regulatory properties of traditional Chinese medicine (especially carbon-based drugs) and the complex pathophysiology of UC, we prepared LLC, which demonstrated excellent hemostatic activity and ROS scavenging capacity (Fig. 2–3), for multi-target intervention in UC. Treatment with LLC effectively alleviated disease symptoms, as evidenced by reduced colonic shortening, mitigated mucosal damage, improved histopathological scoring, and decreased diarrhea and hematochezia (Fig. 4–6). Furthermore, LLC significantly downregulated colonic levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and oxidative stress markers (ROS, MDA, NO) (Fig. 7). Mechanistically, LLC was found to inhibit NLRP3 inflammasome activation, Caspase-1, and GSDMD production, thereby suppressing pyroptosis in colonic epithelial cells (Fig. 8–9). At the systemic immune level, LLC modulated splenic T-cell homeostasis by balancing the CD4 +/CD8 + ratio and the Th17/Treg equilibrium, contributing to the resolution of intestinal inflammation (Fig. 10). Additionally, LLC promoted mucosal healing by upregulating key tight junction proteins (Claudin-1, Occludin and ZO-1) (Fig. 11) and positively reshaped the gut microbiota by enriching beneficial bacteria while suppressing potential pathogens (Fig. 12). Comprehensive safety evaluation, including chronic toxicity, genotoxicity, and biodistribution studies, is necessary before considering therapeutic applications. Although our study demonstrates that LLC showed no acute toxicity in major organs following short-term administration in this study in colonic epithelial cells and tissue organs, future research should systematically conduct pharmacokinetic studies (absorption, distribution, metabolism, excretion) and long-term toxicity assessments for LLC to provide critical data and establish a safety foundation.

LL, a typical "medicinal and edible" material, combines high safety with renewability, and its raw biomass is often regarded as agricultural waste [60]. This study achieves the high-value utilization of this resource. The biological effects of carbon dots are highly dependent on their surface chemistry, particularly the type and abundance of functional groups [61]. Through systematic process optimization, we investigated the structure – antioxidant activity relationship of LLC prepared under different conditions (Fig. S1–S5). XPS and FTIR analyses revealed that LLC prepared at 450 °C for 1 h retained moderate hydrophilic groups such as –OH, –COOH, and –NH₂, whereas the sample obtained at 550 °C showed significant loss of functional groups due to excessive carbonization. These groups serve as key active sites for radical scavenging, directly explaining the stronger DPPH/ABTS⁺ radical‑scavenging capacity of the 450 °C–1 h LLC. In terms of physicochemical properties, the as‑prepared LLC exhibited excellent colloidal stability, with a zeta potential as high as –31.4 mV (Fig. S7)., which is significantly more negative than that of most reported herbal carbon dots (e.g., from Rheum ribes [62] or Eucommia ulmoides charcoal [63]). This high negative charge originates from the abundant oxygen‑containing groups on its surface, which not only ensures uniform dispersion in biological media but may also facilitate intestinal delivery. Functionally, this study is the among the first to report that LLC can synergistically exert multiple effects encompassing hemostasis, anti‑inflammation, mucosal repair, immune regulation, and microbiota remodeling. Notably, LLC not only alleviates inflammation by suppressing NLRP3‑mediated pyroptosis in the colon but also significantly corrects the imbalance in splenic CD4⁺/CD8⁺ T‑cell ratio and the Th17/Treg equilibrium in UC mice. Such systemic modulation of both pyroptosis and adaptive immunity has not been reported in existing studies on herbal carbon dots for UC treatment. However, most descriptions remain at the level of phenomenological correlations. As the most crucial active substance, the molecular interaction mechanism between the surface functional groups of carbon nanodots and specific biological targets (such as receptors or enzymes) remains unexplained.

Intriguingly, LLC treatment specifically enriched five bacterial species in UC mice, including Muribaculaceae_bacterium and Bacteroides acidifaciens (Fig. 12). The increased relative abundance of these beneficial bacteria showed a significant negative correlation with the levels of multiple pro-inflammatory cytokines and key oxidative stress markers in colon tissue (Fig. 7), suggesting a functional link between LLC-induced beneficial remodeling of the gut microbiota and the alleviation of local intestinal inflammation and oxidative stress. Notably, bacteria from the family Muribaculaceae and Bacteroides acidifaciens are important producers of short-chain fatty acids (SCFAs, such as butyrate and propionate) [64, 65]. Butyrate, in particular, is well-documented to exert multiple intestinal protective effects by enhancing barrier function, modulating immune balance (e.g., inhibiting Th17 and promoting Tregs), and activating the Nrf2 pathway to mitigate oxidative stress[66, 67]. In this study, while H-LLC significantly enriched these SCFA-producing bacteria, it also effectively suppressed the expression of pro-inflammatory factors (TNF-α, IL-6, IL-17) and reduced oxidative damage markers (MDA, ROS). This multi-layered, synergistic effect pattern aligns well with the known functions of SCFAs. Therefore, we reasonably speculate that the enrichment of SCFA-producing bacteria coincided with reduced inflammation and oxidative stress, raising the hypothesis that microbial metabolites might participate in LLC’s therapeutic effects. However, direct evidence of SCFA-mediated mechanisms remains to be established.

The 2020 edition of the "Chinese Pharmacopoeia" records that LLC is carbonized using the slow roasting method, where LL are placed in a sealed roasting pot and heated to the desired degree, resulting in LLC appearing as irregular flakes with a brownish-black or blackish-brown surface, a fragrant aroma, and astringent taste [68]. However, this traditional carbonization method is difficult to control in terms of the degree of carbonization and poses operational challenges [27]. To date, new carbonization methods have been developed, including sand heat carbonization, microwave carbonization, muffle furnace calcination, and oven carbonization [11]. The muffle furnace calcination method offers advantages such as ease of operation, good reproducibility, and ease of standardization, and is gradually being adopted as an improved method for preparing carbon medicines, such as the preparation of Scutellaria baicalensis carbon [69] and LLC [70]. Research has shown that LLC prepared by calcining in a muffle furnace at 250, 350, and 450 °C for 1 h exhibited the best hemostatic effect at 450 °C [30]. Based on this, our experiments also used this condition to prepare LLC. During the experimental process, we found that LLC calcined at 450 °C for 1 h demonstrated excellent performance in hemostasis, anti-inflammation, and antioxidant activities. In the future, further exploration of the effects of different calcination conditions on the physiological activities of LLC can provide more scientific evidence for the in-depth study of LLC.

Since ancient China, the theory of ‘blood stops when it sees black’ has deeply resonated within society. Current academic research on the hemostatic substance basis of carbon medicines primarily focuses on the significantly changed chemical components before and after processing, such as carbon, tannins, calcium ions, and organic compounds (flavonoids, anthraquinones, saponins, etc.) [71]. However, there is a lack of reasonable and unified explanations for why various traditional Chinese medicines, which have different effects, exhibit a certain hemostatic function after high-temperature carbonization [72]. It is noteworthy that the high-temperature carbonization process of carbon medicines is quite similar to the high-temperature pyrolysis method used in modern nanomaterials known as ‘carbon dots’: raw materials (herbs/carbon sources) are placed in a crucible and heated to complete carbonization at specific temperatures [73]. This commonality in processing indirectly supports the hypothesis that carbon medicines may inherently contain carbon dots. In fact, modern research has also identified carbon dots with hemostatic effects from Carbonized Platycladus [39], Gardenia charcoal [74], and Cirsii Japonici Herba Carbonisata [75], revealing that the hemostatic substance basis of carbon medicines may be closely related to the nano-scale carbon structures formed during high-temperature carbonization. Therefore, we propose that one of the important active substance bases in carbon medicines is carbon dots. In our study, we also discovered carbon dots from LLC, and in vitro experiments showed that LLC has excellent pro-coagulation effects; in UC mice, LLC was found to reduce hematochezia and shorten tail bleeding time, thereby decreasing the amount of bleeding. Based on the correlation between carbonization conditions, nanoparticle formation, and enhanced hemostatic activity, we hypothesize that carbon dots may partially account for the ‘charring for hemostasis’ principle in traditional medicine. However, this interpretation remains speculative without direct evidence from component-depletion experiments.

Carbon dots, as a type of nanomaterial, exhibit various biological activities such as anti-inflammatory and antioxidant effects. Research has shown that honeysuckle-derived carbon dots can alleviate lipopolysaccharide-induced fever by reducing the levels of inflammatory factors, thereby exerting anti-inflammatory effects [25]. Licorice-derived carbon dots can mitigate the damage to gastric mucosa caused by free radicals during alcohol metabolism by enhancing the antioxidant capacity in a mouse model of gastric ulcers [76]. This study applies LLC in the treatment of UC and for the first time identifies carbon dots may represent an important component of the material basis for LLC in UC therapy. This finding challenges the traditional view that the efficacy of LLC relies on its existing alkaloid and flavonoid components, opening new avenues for research in the field of carbon-based medicines, including LLC. However, LLC is a complex mixture that may contain other carbonization products or residual inorganic components besides carbon dots. While it is reasonable to attribute the therapeutic effects primarily to carbon dots, more controlled experiments (such as comparing purified carbon dot components with the whole extract) are needed to provide stronger evidence. Through interdisciplinary collaboration and the integration of advanced techniques such as modern nanotechnology and bioinformatics, there is potential to accelerate the modernization of carbon-based medicines and promote a deeper integration of traditional Chinese medicine with modern technology.

Conclusion

In this study, LL, a medicinal and edible material, was used as a precursor to prepare LLC via high‑temperature pyrolysis, and its therapeutic potential in DSS‑induced UC was evaluated. Carbon‑based nanoparticles were found to be uniformly dispersed in the LLC system. Both in vitro and in vivo experiments demonstrated that LLC possesses notable hemostatic and antioxidant activities. In the UC mouse model, LLC exhibited multi‑faceted regulatory effects, including mitigating inflammatory responses, suppressing pyroptosis, modulating local immune status, enhancing intestinal barrier function, and reshaping gut microbiota composition, with an overall efficacy superior to that of raw LL. These findings suggest that the carbon‑based nanostructures formed during LLC preparation may contribute to its biological effects, providing a novel scientific interpretation for the traditional theory of “charring for hemostasis.” While carbon dots represent a promising candidate for the hemostatic and anti-inflammatory properties of LLC, we acknowledge that the whole extract contains multiple components that may act synergistically. In conclusion, as a natural product‑derived carbon material, LLC shows promising therapeutic potential for UC and warrants further investigation. Although LLC demonstrated efficacy in a murine DSS-induced colitis model, translation to human applications requires comprehensive pharmacokinetic profiling, long-term toxicity assessment, and validation in more complex disease models before clinical consideration.

Supplementary Information

Additional file 1^{(21.6MB, docx)}

Acknowledgements

We acknowledge the support of experiment center for science and technology at Nanjing University of Chinese Medicine for technical support; Jiangsu key laboratory of Chinese Medicine Processing for experimental support. Mechanistic cartoons were created by BioRender.com.Thank Chenchen Lin from SCI-GO (www.SCI-go. com) for the FE-SEM analysis.

Author contributions

Yu Zheng: Data curation, Investigation, Formal analysis, Validation, Writing-original draft; Jiangcui Liu: Formal analysis, Data curation, Methodology; Ying Ma: Investigation, Validation; Yunyu Zhang: Resources; Heng Liu: Supervision; Rui Chen: Conceptualization, Supervision, Funding acquisition, Writing-review & editing. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

Supported by National Natural Science Foundation (No. 82474195) and Yunnan Province Young and Middle aged Academic and Technical Leaders Reserve Talent Project (No. 202305AC160034).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Yu Zheng and Jiangcui Liu contributed equally to this work.

Contributor Information

Heng Liu, Email: lheng125@dali.edu.cn.

Rui Chen, Email: chenrui@njucm.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional file 1^{(21.6MB, docx)}

Data Availability Statement

No datasets were generated or analysed during the current study.

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PERMALINK

Calcined lotus leaf-derived carbon dots: enhanced hemostasis, anti-inflammatory, immunomodulatory properties for ulcerative colitis management

Yu Zheng

Jiangcui Liu

Ying Ma

Yunyu Zhang

Heng Liu

Rui Chen

Abstract

Graphical Abstract

Supplementary Information

Introduction

Scheme 1.

Materials and methods

Materials

Preparation of LL and LLC extracts

Structural characterization

Calculation of the carbonization yield (CY)

Measurement of the quantum yield (QY)

In vitro inverted coagulation experiment

Coagulation time determination

Hemodynamic measurement

Analysis of Platelet Count and Coagulation Function

Free radical scavenging experiment

In Vivo imaging of gastrointestinal accumulation

Modeling and treatment of UC

General observations of physical characteristics

DAI scores

Table 1.

Calculation of Organ Index

Measurement of Macroscopic indicators related to the colon

Table 2.

H&E staining, PAS staining, and histopathological scoring

Table 3.

Tail amputation experiment measured bleeding time and blood volume

Detection of cytokine levels by ELISA

Antioxidant measurement

CCD-841CoN cell culture and CCK-8 assay

Detection of p-NF-κB p65, NLRP3, caspase-1 and ASC mRNA expression in CCD-841CoN cells by RT-qPCR

Western blot detection of NLRP3/caspase-1/GSDMD protein expression in colon tissue

Immunofluorescence Detection of NLRP3, caspase-1, GSDMD and ASC protein expression in mouse colon tissue

Detection of CD3+/CD4+/CD8+ T lymphocyte levels and cytokine levels of IL-17 and treg in the spleen and lymph nodes by flow cytometry

Immunofluorescence staining of Claudin-1, occludin and ZO-1

Analysis of gut microbiota

Statistical Methods

Results

Discovery and characterization of CDs in LLC

Fig. 1.

Hemostatic effect of LLC

Fig. 2.

Antioxidant activity and gastrointestinal retention capacity of LLC

Fig. 3.

LLC alleviates UC

Fig. 4.

LLC alleviates pathological damage in mice with UC

Fig. 5.

Hemostatic properties of LLC

Fig. 6.

In Vivo anti-inflammatory and antioxidant effects of LLC

Fig. 7.

Effects of LLC on NLRP3/caspase-1/GSDMD-mediated pyroptosis pathway

Fig. 8.

Fig. 9.

Regulation of immune homeostasis by LLC in mice with UC

Fig. 10.

LLC Restores intestinal barrier function

Fig. 11.

Regulation of gut Microbiota by LLC

Fig. 12.

Discussion

Conclusion

Supplementary Information

Acknowledgements

Author contributions

Funding

Data availability

Declarations

Competing interests

Footnotes

Contributor Information

Detection of CD3⁺/CD4⁺/CD8⁺ T lymphocyte levels and cytokine levels of IL-17 and treg in the spleen and lymph nodes by flow cytometry