Alfalfa Flavonoids Mitigate Salmonella‐Induced Colitis via the Keap1‐Nrf2 and TLR4/NF‐κB/COX‐2 Pathways

Abstract

ABSTRACT

Alfalfa is rich in flavonoid compounds, which are known for their antioxidative and anti‐inflammatory properties, suggesting therapeutic potential for alfalfa flavonoids (AF) in inflammation‐related diseases. This study investigated the effects of AF on Salmonella‐induced colitis, a severe inflammatory bowel disorder characterized by oxidative damage and inflammatory response. In vitro, antioxidant assays revealed AF's concentration‐dependent radical scavenging, significantly reducing electron paramagnetic resonance (EPR) signals for HO^• and O₂^•− by 42% and 54%, respectively. In vivo, AF treatment significantly mitigated body weight (BW) loss by 6%, increased colon length by 11%, and reduced liver and spleen weights by 19% and 81%, respectively, compared to the colitis group. Mechanistically, AF suppressed inflammation by downregulating the Toll‐like receptor 4 (TLR4)/IκB/nuclear factor‐κB (NF‐κB)/cyclooxygenase‐2 (COX‐2) pathway and inhibiting nucleotide‐binding domain‐like receptor protein 3 (NLRP3) inflammasome activation, thereby lowering levels of pro‐inflammatory cytokines (tumor necrosis factor alpha [TNF‐α], interleukin 6 [IL‐6], interleukin 1 beta [IL‐1β]). Concurrently, AF enhanced antioxidant defense via the Kelch‐like ECH‐associated protein 1 (Keap1)‐nuclear factor erythroid 2‐related factor 2 (Nrf2) pathway, reducing reactive oxygen species (ROS) and malondialdehyde (MDA) levels while increasing catalase (CAT) (69%), glutathione peroxidase (GPx) (83%), and superoxide dismutase (SOD) (21%) activities. Moreover, AF preserved epithelial and mucosal barriers by reducing apoptosis and upregulating tight junction proteins (Claudin1, ZO‐1, E‐cadherin) and goblet cell marker Ulex europaeus (Gorse) Agglutinin I (UEA‐1). Microbiota analysis revealed that AF significantly enriched beneficial bacteria, including Akkermansia, Oscillibacter, and butyrate‐producing taxa, thereby counteracting Salmonella‐induced dysbiosis. Furthermore, AF restored the disrupted profile of short‐chain fatty acids (SCFAs), strengthening the relationship between symbiotic microbiota and mucosal defense. Overall, AF exerted multifaced protection against Salmonella‐induced colitis by alleviating oxidative stress, stabilizing intestinal homeostasis, and thus attenuating inflammation. These findings make AF a promising phytopharmaceutical for the prevention and treatment of inflammatory diseases.

Abbreviations

• AF
• alfalfa flavonoids
• CAT
• catalase
• COX-2
• cyclooxygenase-2
• EPR
• electron paramagnetic resonance
• GPx
• glutathione peroxidase
• Keap1
• Kelch-like ECH-associated protein 1
• MDA
• malondialdehyde
• NF-κB
• nuclear factor-κB
• Nrf2
• nuclear factor erythroid 2-related factor 2
• ROS
• reactive oxygen species
• SCFAs
• short-chain fatty acids
• SOD
• superoxide dismutase
• T-AOC
• total antioxidant capacity
• TLR4
• Toll-like receptor 4

Introduction

Salmonella, a major foodborne pathogen, causes approximately 129.5 million cases of gastroenteritis and 155,000 deaths annually, resulting in significant global health and socioeconomic burdens (Majowicz et al. 2010; Branchu et al.2018). Salmonella can survive within phagocytes and induce intestinal damage, often leading to inflammatory bowel disease characterized by severe diarrhea and microbial dysbiosis. This inflammatory response is triggered when intestinal epithelial cells recognize pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides (LPS) through Toll-like receptors (TLRs) (Swanson et al. 2019). TLR4 is among the most crucial TLRs involved in regulating inflammation. When stimulated by LPS, TLR4 activates nuclear factor-κB (NF-κB) by binding to the adaptor protein myeloid differentiation factor 88 (MyD88), which promotes the expression of pro-inflammatory cytokines (He et al. 2021). C. Y. Chen et al. (2018) reviewed a variety of bioactive phytochemicals that reduced inflammation by inhibiting TLR4 activation. However, there are currently no reports on alfalfa flavonoids (AF) on the molecular events of the inflammatory process. Additionally, Salmonella-induced inflammation is often exacerbated by oxidative stress, which results from an imbalance between reactive oxygen species (ROS) production and antioxidant defenses (Bhattacharyya et al. 2014). The Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway plays an important role in resisting oxidative stress (Dong et al. 2024; Lv et al. 2025). Keap1 is a ROS sensor that monitors the redox state of the cell and communicates this information to Nrf2 (Sykiotis et al. 2021). Nrf2 is a stress-responsive transcription factor that is essential for cellular homeostasis. It detects oxidative stress and triggers the expression of related antioxidant genes. Research has demonstrated that TLR4/MyD88 signaling can activate the Nrf2 pathway (Morsy et al. 2022). Furthermore, the Nrf2/heme oxygenase-1 (HO-1) pathway has been shown a vital role in preventing inflammatory bowel disease (Bourgonje et al. 2023). However, the role and mechanism of AF in modulating the Keap1/Nrf2 pathway and the interaction with inflammatory regulators remains unclear.

Phytopharmaceuticals have garnered increasing attention for their ability to modulate inflammatory responses without the side effects of conventional drugs, making them a promising alternative for treating foodborne pathogens. Alfalfa (Medicago sativa L.), known as the “king of forages,” has long been recognized for its nutritional value, including proteins, minerals, and vitamins. Widely employed in grass-fed livestock, alfalfa has been verified to boost milk production and augment daily weight gain in cattle (Broderick et al. 2002; Crump et al. 2024). Beyond its established nutritional benefits and role in enhancing livestock productivity, alfalfa contains bioactive compounds, notably flavonoids (AF), which exhibit potent antioxidant properties, suggesting their potential as a therapeutic option for inflammation-related diseases such as Salmonella-induced colitis. In 2009, the European Food Safety Authority approved alfalfa leaf extract as a dietary supplement (Bresson et al. 2009). Previous studies have focused on the production performance and antioxidant properties of AF. For example, AF has shown potential growth-promoting effects in sheep (M. Wang et al. 2015). Similarly, adding AF extract to the diet promoted the growth performance of Yangzhou geese (Y. Chen et al. 2016). Additionally, supplementing chickens with AF promoted average daily weight gain and meat quality and enhanced antioxidant activity (Ouyang et al. 2016; Zhan et al. 2017). Moreover, supplementation of the AF also improved antioxidant properties in cattle (Zhan et al. 2017) and in rabbits (Dabbou et al. 2018). A recent study has also highlighted their ability to modulate the intestinal microbiota (Xie et al. 2023). However, these studies did not further explore the potential mechanisms of its positive response to production performance and antioxidant properties.

We hypothesize that AF could prevent and alleviate inflammation in Salmonella-induced colitis by modulating antioxidant signaling, anti-inflammation responses, and gut microbiota. In this study, we investigated for the first time the mechanism underlying the prevention and therapeutic roles in Salmonella-induced colitis by AF and its regulatory effects on maintaining intestinal barrier integrity in a mouse model. To assess the antioxidant capacity of AF treatment, in vitro antioxidant and electron paramagnetic resonance (EPR) assays were performed to evaluate its radical-scavenging capacity. In vivo, the antioxidant and anti-inflammatory mechanisms of AF were further investigated by examining the NF-κB/cyclooxygenase-2 (COX2) and Nrf2/HO-1 pathways using RT-PCR and Western blotting techniques, with immunofluorescence staining providing additional validation. Furthermore, 16S rDNA sequencing was employed to identify the differential bacteria induced by AF treatment. This study provides a comprehensive investigation into the ability of AF to prevent and mitigate Salmonella-induced colitis symptoms by the modulation of oxidative stress, inflammation, intestinal barrier function, and microbiota. The findings would support the potential of AF as a safe and effective herbal medicine for the prevention and treatment of inflammatory diseases.

Materials and Methods

Chemicals

AF were purchased from Shaanxi Green Bio-Engineering Co. Ltd. (Shaanxi, China); 2,2-diphenyl-1-picrylhydrazyl radical (DPPH, ≥97%, D273092), riboflavin (≥98%, R104137), 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO, ≥98%, P160514), 2,3,5-triphenyltetrazolium chloride (TPTZ, ≥99%, T106623), methylene blue (MB, ≥70%, M134389), nitro blue tetrazolium chloride (NBT, ≥98%, N104908), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, ≥98%, A109612), 2,4,6-trinitrobenzene sulfonic acid chloride (TNBS, ≥99%, T106623), sodium acetate trihydrate (≥99%, S111519), potassium peroxodisulfate (FeSO₄·7H₂O, P433869), hydrogen peroxide (H₂O₂, 30%, H433859), iron(II) sulfate heptahydrate (≥98%, I434044), and iron chloride hexahydrate (FeCl₃, ≥98%, F419646) were acquired from Aladdin Scientific Corp. in Shanghai, China.

Quantification of Flavonoids by LC–MS/MS

AF samples were identified and quantified using LC–MS/MS as previously described (Leoni et al. 2021). The AF solutions were separated by an ultrahigh-performance liquid chromatography system (Agilent 1290 Infinity LC, California, USA) equipped with ACQUITY UPLC HSS T3 Column (1.8 µm, 2.1 mm × 100 mm, Waters, MA, USA). The mobile phase A consisted of 0.1% formic acid (≥98.00%, 94318, Honeywell, NJ, USA) in water, and the mobile phase B contained 0.1% formic acid in acetonitrile (≥99.9%, A9554, Thermo Fisher Scientific, MA, USA). The flow rate was set at 400 µL/min, and the injection volume was 2 µL. The liquid phase gradient was as follows: phase B increased linearly from 5% to 20% in 0–3 min; phase B increased linearly from 20% to 45% in 3–9 min; phase B increased linearly from 45% to 95% in 9–11 min; phase B was maintained at 95% in 11–13 min; phase B decreased linearly from 95% to 10% in 13–13.1 min, and phase B was maintained at 10% in 13.1–15 min. The mass spectrometry analysis was performed by a 5500 QTRAP mass spectrometer (SCIEX, USA) in positive/negative ion mode. The electrospray ionization (ESI) source positive ion conditions were as follows: source temperature, 550°C; ion source Gas1 (Gas1), 55 psi; ion source Gas2 (Gas2), 50 psi; curtain gas (CUR), 30 psi; ionSapary Voltage Floating (ISVF), 5500 V. The ESI source negative ion conditions were as follows: source temperature, 550°C; Gas1, 55 psi; Ion Gas2, 50 psi; CUR, 30 psi; ISVF, −4500 V. The multiple reaction monitoring (MRM) mode detected the ions to be measured and quantified.

Scavenging Ability Against DPPH^• of AF

DPPH^• is a radical known for its hydrogen-accepting abilities as an antioxidant (Sheng et al. 2022). To prepare a 1 mM storage solution (10×), 50 mg of DPPH is first dissolved in 125 mL of anhydrous ethanol. Then, 375 µL of AF solution (A) or water (A₀) is mixed with 750 µL of the working solution (1×). After the solution darkens for half an hour at room temperature, the BioTek Synergy LX (BioTek, USA) is used to measure the absorbance at 517 nm: $$$\begin{equation*}{\mathrm{Scavenging\;rate\;}}\left( {\mathrm{\% }} \right) = \left[ {\left( {{{\mathrm A}_0} - {\mathrm A}} \right)/{{\mathrm A}_0}} \right] \times 100{\mathrm{\% }},\end{equation*}$$$where A₀ represents the control absorbance, and A represents the absorbance of AF solution.

Scavenging Ability Against PTIO^• of AF

The scavenging capacity of PTIO^•, which exhibits characteristic absorbance at 557 nm, is evaluated by its decolorization. A volume of 200 µL of AF solution at various concentrations is added to 1.8 mL of 50 µg/mL PTIO: $$$\begin{equation*}{\mathrm{Scavenging\;rate\;}}\left( {\mathrm{\% }} \right) = \left[ {\left( {{{\mathrm A}_0} - {\mathrm A}} \right)/{{\mathrm A}_0}} \right] \times 100{\mathrm{\% }},\end{equation*}$$$where A₀ represents the absorbance of the reference substance, and A represents the mixture's absorbance.

Scavenging Ability Against ABTS^•+ of AF

A mixture of 7 mM ABTS and 2.45 mM potassium persulfate in a 100 mL solution is allowed to sit overnight in the dark. Subsequently, 1 mL of the mixture is added to 100 mL of anhydrous ethanol and diluted to an absorbance of 0.7 ± 0.02. Next, 3.9 mL of this ABTS solution is combined with 100 µL of varying concentrations of AF solution (A) or ethanol (A₀). The optical density at 734 nm is determined following a half-hour reaction in the absence of light exposure: $$$\begin{equation*}{\mathrm{Scavenging\;rate\;}}\left( {\mathrm{\% }} \right) = \left[ {\left( {{{\mathrm A}_0} - {\mathrm A}} \right)/{{\mathrm A}_0}} \right] \times 100{\mathrm{\% }}.\end{equation*}$$$

Ferric-Reducing Antioxidant Power Assay (FRAP) of AF

A 10 mM TPTZ, 20 mM FeCl₃ solution, and 3 M sodium acetate buffer were mixed at the ratio of 10:1:1 at pH 3.6. Add 0.15 mL water (A₀) or AF solution (A) at various concentrations to 4.5 mL of FRAP solution preheated to 37°C, incubate in darkness for 30 min, and then conduct a spectral scan using the BioTek Synergy LX (BioTek, USA) and measure the absorbance at 593 nm: $$$\begin{equation*}{\mathrm{Scavenging\;rate\;}}\left( {\mathrm{\% }} \right) = \left[ {\left( {{{\mathrm A}_0} - {\mathrm A}} \right)/{{\mathrm A}_0}} \right] \times 100{\mathrm{\% }}.\end{equation*}$$$

Scavenging Ability Against Hydroxyl Radical (HO^•) of AF

A solution containing HO^• was prepared through a Fenton reaction using FeSO₄·7H₂O (40 mM) and H₂O₂ (80 mM). This reaction oxidizes MB (200 µg/mL) to its oxidized state (oxMB), decreasing the absorbance at 665 nm. The spectral scanning was conducted using the BioTek Synergy LX (BioTek, USA): $$$\begin{equation*}{\mathrm{Scavenging\;rate\;}}\left( {\mathrm{\% }} \right) = \left[ {\left( {{\mathrm A} - {{\mathrm A}_0}} \right)/\left( {{A_{{\mathrm{MB}}}} - {{\mathrm A}_0}} \right)} \right] \times 100{\mathrm{\% }},\end{equation*}$$$where A₀ is the absorbance of the control, A is the absorbance of the post-reaction AF solution, and A_MB is the absorbance of the pure MB solution.

Scavenging Ability Against O₂^•− of AF

Riboflavin, as an electron donor, produces superoxide anion radical (O₂^•−) when illuminated in methionine. This superoxide reduces NBT to form blue formazan, which exhibits characteristic absorption at 560 nm. A mixture comprising various concentrations of PBS or AF solution, riboflavin (20 µm), methionine (130 mM), NBT (750 µm), and EDTANa₂ (100 µm) was irradiated under 4000 lux for 20 min, followed by a complete spectral scan to assess the reduction of NBT: $$$\begin{equation*}{\mathrm{Scavenging\;rate\;}}\left( {\mathrm{\% }} \right) = \left[ {\left( {{{\mathrm A}_0} - {\mathrm A}} \right)/{{\mathrm A}_0}} \right] \times 100{\mathrm{\% }},\end{equation*}$$$where A₀ represents the absorbance of PBS, and A represents the AF solution's absorbance.

EPR Measurement

Using DMPO as a spin-trapping agent, the scavenging abilities of HO^• and O₂^•− were studied using EPR. First, a solution containing 500 mM H₂O₂ and 100 mM DMPO was exposed to UV light for 5 min. Subsequently, EPR signals were collected both in the presence and absence of AF to estimate the scavenging abilities of HO^• and O₂^•−.

Animal Experiments

All experiments adhered to protocols approved by the Animal Protection and Ethics Committee of China Agricultural University (AW29093202-1-2). The mice were housed in SPF facilities with free access to food and water.

Forty-eight male mice, aged 6 weeks, were acquired from Vital River Laboratory Animal Technology Co. Ltd. in Beijing, China, and maintained on a regular diet. As depicted in Figure 2A, the mice were randomly divided into six groups: control (CON), Salmonella-infected (SALM), two AF-treated groups receiving 40 or 120 mg/kg body weight (BW) d of AF (AF40 and AF120), and two combined treatment groups with Salmonella infection and AF supplementation (SALM + AF40 and SALM + AF120). The dosages of AF used in this study were based on previous research investigating the effects of alfalfa-derived flavonoids (S. Chen et al. 2020; Dabbou et al. 2018; Ouyang et al. 2016; Zhan et al. 2017). The dosage of 40 mg/kg BW d represents a modest supplementation with possible therapeutic benefits, whereas the dosage of 120 mg/kg BW d was included to examine the maximum protective effects of AF. The mice were given daily oral doses of AF or an equivalent volume of PBS from 4 weeks before Salmonella infection until 8 days after infection. A volume of 200 µL of 1 × 10⁹ CFU/mL Salmonella typhimurium strain SL1344 or PBS was given to the mice. Eight days after infection, all mice were anesthetized, and samples of blood, liver, and colon were collected for further analysis.

Pathological Observations of Colon

The colon samples were sectioned at 5 µm and stained with hematoxylin and eosin (HE). The pathological scoring was carried out according to the established criteria (He et al. 2021). Alcian blue was used to stain the acidic mucopolysaccharides secreted by goblet cells, and the ratio of stained positive areas within the intestinal epithelium was analyzed using ImageJ software.

Oxidative Stress Analysis

The activities of total antioxidant capacity (T-AOC) (Cat. No. A015), superoxide dismutase (SOD) (Cat. No. A001-1), catalase (CAT) (Cat. No. A007-1), glutathione peroxidase (GPx) (Cat. No. A005), as well as the level of malondialdehyde (MDA) (Cat. No. A003-1) in liver homogenates, were measured using kits from Jiancheng Bioengineering Institute (Nanjing, China).

Cytokine Assays

The concentrations of tumor necrosis factor alpha (TNF-α) (Cat. No. mIC50536-1), interleukin 1 beta (IL-1β) (Cat. No. mIC50300-1), and interleukin 6 (IL-6) (Cat. No. mIC50325-1) in serum were tested using ELISA kits obtained from Shanghai Enzyme-linked Biotechnology in Shanghai, China.

RNA Extraction and Real-Time Quantitative PCR (RT-PCR)

The total RNA of the colon tissue was isolated with TRIzol reagent (P118-05, GenStar, Beijing, China). The StarScript III First Strand cDNA Synthesis Kit (A230-10, GenStar, Beijing, China) was utilized to synthesize cDNA. RT-PCR was conducted in a 20 µL reaction system (A301-10, GenStar, Beijing, China). The reaction protocol was begun at 95°C for 5 min, succeeded by 40 cycles of 95°C for 10 s, 60°C for 30 s, 72°C for 30 s, concluding with a final extension at 72°C for 10 min. Analysis of the RT-PCR results was conducted utilizing the 2^−ΔΔCT technique, with β-actin serving as the reference gene. Primer sequences are listed in Table S2.

Western Blot

The method of Western blot was referred to in the previous study (Qin et al. 2025). Proteins from colon tissue were lysed and extracted with RIPA lysis buffer (P0013B) that included protease and phosphatase inhibitors from Beyotime in Shanghai, China. The protein concentration was measured with an improved BCA protein assay kit (P0009, Beyotime, Shanghai, China). An appropriate amount of concentrated SDS–PAGE sample buffer was mixed with the protein samples, followed by heating in a boiling water bath for 10 min to fully denature the proteins. After being separated on an 8%–12% SDS-polyacrylamide gel, the proteins were then transferred onto a PVDF membrane. After transfer, the protein was blocked with Western blocking solution at room temperature for 60 min and then incubated with primary antibodies overnight at 4°C. The membrane was washed with TBST buffer and then incubated for 1 h with an appropriate secondary antibody. The ChemiDoc imaging system (Bio-Rad, USA) was utilized for band identification, followed by quantification using ImageJ software (NIH, USA) with normalization of protein band density to tubulin expression. Antibody information is listed in Table S3.

Immunofluorescence

Colon samples were embedded with OTC compound and sectioned into 7 µm thick frozen sections. The cut sections were immersed in citric acid solution (50 mM) for 10 min at 100°C in a water bath for antigen recovery. The sections were subsequently permeabilized with 0.4% Triton-100 for 30 min and incubated with a 5% bovine serum albumin solution in PBS for 1 h. Then, the tissues were left overnight with primary antibodies at 4°C, rinsed with PBS, treated with Hoechst (1:5000) for 5 min, rinsed once more, and ultimately covered with an anti-fluorescence quenching mounting medium. Two hours before euthanasia, mice were given an injection of 40 mg/kg of 5-ethynyl-2′-deoxyuridine (EdU) to identify proliferating cells using the BeyoClick EdU Cell Proliferation Kit (C0071S, Beyotime, Shanghai, China). Apoptosis was detected using the TUNEL assay (C1088, Beyotime, Shanghai, China), which specifically evaluated nuclear DNA fragmentation with an apoptosis detection kit from Beyotime in Shanghai, China. Goblet cells were identified using FITC-conjugated Ulex europaeus (Gorse) Agglutinin I (UEA-1) staining. ROS were determined by staining frozen colon sections in the colon with 10 µM DCFH-DA (M36008, Invitrogen, USA) for 30 min before images were acquired with a confocal microscope (Leica TCS SP8 confocal laser microscope, Mannheim, Germany). Antibody information is listed in Table S3.

Short-Chain Fatty Acids (SCFAs) Assay

Fecal SCFAs concentrations were determined using a gas chromatograph (Shimadzu GC-2014, Kyoto, Japan), equipped with a 30-m capillary column (Rtx-Wax, 0.25-mm i.d., 0.25-µm film thickness, Restek, Evry, France) and a hydrogen flame detector. The internal standard, 2-ethylbutyric acid (≥99.5%, E105669), was purchased from Aladdin (Shanghai, China). The instrument was operated with the following parameters: nitrogen flow rate of 46.3 cm/s, injection volume of 0.4 µL, and injection port temperature set to 220°C. The heating program was as follows: The initial column temperature was set at 110°C for 30 s and then raised to 120°C at a rate of 10°C/min, where it was maintained for 4 min. Finally, the temperature was increased to 150°C at a rate of 10°C/min, and the detector temperature was set at 250°C.

Fecal Microbiota 16S rDNA Sequencing

DNA from fecal samples was isolated with the E.Z.N.A. DNA extraction kit for stool samples (D2500-00, Omega Biotek Inc., USA). Primers 338F and 806R were used to amplify the V3–V4 regions of the bacterial 16S rRNA gene for sample differentiation. PCR amplification was conducted on an ABI 9700 PCR system (Applied Biosystems Inc., USA). Sequencing was performed on an Illumina MiSeq platform (San Diego, CA, USA) following the provided protocols to produce paired-end reads of 250 base pairs. The PCR products were processed for library construction with the kit (New England Biolabs Inc., USA). Sequencing data were processed and analyzed following our previously established methods (Qin et al. 2022).

Statistical Analysis

The findings were depicted as mean ± standard deviation (SD). ANOVA test was chosen to compare significant differences between groups using GraphPad Prism 9. Significant distinctions were indicated by *p < 0.05, **p < 0.01, ***p < 0.001.

Results

ROS Scavenging Activities of AF

On the basis of LC–MS/MS analysis, AF is identified as a mixture of multiple flavonoid components, for example, quercetin (296.0 mg/g), isorhamnetin (57.0 mg/g), quercetin-3-glucoside (42.0 mg/g), rutin (12.3 mg/g), genistein (8.2 mg/g), and apigenin (7.2 mg/g) (Figure 1, Table S1). These compounds have typical structural characteristics of flavonoids, including a flavonoid backbone consisting of an aromatic ring (A, six-membered aromatic ring-benzene ring) and a phenyl ring (B, attached to the C2 position of the C ring via a single bond) and a heterocyclic ring (C, heterocyclic six-membered ring containing one oxygen atom, forming a γ-pyrone structure), chemically. The antioxidant properties of AF are primarily attributed to its unique chemical structure, such as the B-ring catechol groups, the C-ring conjugation (C2 = C3 and C4 = O), and the A-ring hydroxyl groups, which work synergistically to donate electrons to neutralize oxidative stress for protecting biological systems.

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The ROS scavenging activities of AF were evaluated in vitro against HO^•, O₂^•−, DPPH^•, ABTS^•+, PTIO^•, and FRAP. Initially, we assessed AF's scavenging performance against O₂^•− one of the most destructive ROS. In this assay, l-methionine acts as an electron donor to reduce riboflavin, generating O₂^•− under photosensitization. Nitro-blue tetrazolium chloride then reacts with O₂^•− to form blue formazan, which absorbs at 560 nm. By quantifying the remaining formazan, we determined that AF's O₂^•− scavenging capability increased linearly with its concentration, as evidenced by the progressive fading of the mixture's color. Subsequently, we evaluated AF's scavenging performance against HO^•. The addition of AF effectively scavenged HO^•, reducing MB oxidation and slowing its fading reaction. PTIO^• exhibited significant decolorization and a decline in absorbance at 557 nm upon reaction with AF. As shown in Figure 1B, AF's PTIO^• scavenging ability was strongly concentration dependent. Additionally, DPPH^• and ABTS^•+ assays were employed to evaluate AF's scavenging capacity for nitrogen-centered radicals, revealing that its scavenging effects on both DPPH^• and ABTS^•+ intensified with increasing AF concentration. Moreover, we assessed AF's ferric ion-reducing capability using the FRAP method. Under acidic conditions, TPTZ reacts with Fe³⁺ to create a compound, which is then reduced to Fe²⁺ by AF, resulting in a blue color. The reducing ability of AF for Fe³⁺ was found to increase with its concentration (Figure 1B). Furthermore, EPR spectroscopy with DMPO as a spin-trapping agent was used to detect signals from HO^• and O₂^•−. The detected EPR signals for HO^• and O₂^•− were significantly reduced by 42% and 54%, respectively, by the addition of AF, whereas AF itself produced no EPR signals, reinforcing its role as an effective ROS scavenger (Figure 1C,D). Collectively, these results highlight AF's broad-spectrum ROS scavenging capability.

AF Protected Against the Salmonella-Induced Colitis in Mice

To evaluate the prophylactic and therapeutic effects of AF on Salmonella-induced colitis, mice received 40 mg/kg or 120 mg/kg of AF by gavage continuously from 4 weeks before Salmonella infection until 8 days after infection (Figure 2A). The results demonstrated that mice treated with Salmonella exhibited clear signs of colitis symptoms, including weight loss (Figure 2B), decreased colon length (Figure 2C), increased weight in the liver (Figure 2D) and spleen (Figure 2E), and elevated clinical ratings compared to the control group mice. Compared to the colitis group, SALM + AF40 treatment significantly overcame weight loss (94.1% vs. 87.8%), restored colon length (increased by 11%), and decreased liver (decreased by 19%) and spleen (decreased by 81%) weight. Pathological analysis revealed that 40 mg/kg of AF could alleviate the histological characteristics of colitis, such as inflammatory cell infiltration and mucosal damage (Figure 2F). Furthermore, Alcian blue staining illustrated that the Salmonella treatment group had significantly fewer acidic mucin-positive cells as compared to the control group. Gavage with 40 mg/kg AF alleviated the reduction in acidic mucin secretion caused by Salmonella (Figure 5A). However, mice treated with 120 mg/kg AF did not show significant alleviation of colitis symptoms, as evidenced by the lack of significant reduction in liver and spleen weight and improvement in neutral mucin secretion compared to Salmonella treatment. These research observations collectively indicated that AF, particularly at 40 mg/kg, effectively prevented and relieved the Salmonella-induced colitis symptoms.

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AF against Inflammation via TLR4/IκB/NF-κB/COX-2 Signaling Pathway

Subsequently, the influence of AF on the colonic inflammatory response to Salmonella was further studied. As shown in Figure 3A,B, Salmonella significantly increased the levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in both serum and liver, indicating the systemic inflammatory response and gut–liver axis involvement typical of Salmonella-induced colitis. However, SALM + AF40 effectively suppressed Salmonella’s stimulatory action and curtailed the elevation of pro-inflammatory cytokines. Consistent with the above observation, AF significantly reduced NF-κB and IκB proteins’ phosphorylated levels in the colons of mice subjected to Salmonella, leading to concomitant reductions in the levels of the downstream proteins inducible nitric oxide synthase (iNOS) and COX-2, as depicted in Figure 3D. Moreover, Salmonella treatment resulted in an increased expression level of nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasomes in comparison with the control group. Similarly, the expression levels of Caspase-1 and apoptosis-associated speck-like protein containing caspase-recruitment domain (CARD) (ASC) proteins were observed to be higher in the Salmonella group compared to the control group. In a reverse effect, SALM + AF40 significantly reduced the expression of these proteins induced by Salmonella (Figure 3D). Supporting these protein-related results, SALM + AF40 reduced the increased mRNA expression levels of the NF-κB gene and its upstream genes, TLR4 and MyD88, as well as the COX-2 gene and its downstream gene, CCL2, induced by Salmonella (Figure 3C). Additionally, SALM + AF40 also significantly ablated the Salmonella-enhanced mRNA levels of NLRP3, iNOS, IL-1β, and IL-6 (Figure 3C). Taken together, the anti-inflammatory efficacy of AF predominantly manifests through the attenuation of TLR4/IκB/NF-κB/COX-2 pathway activation.

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AF Attenuated Oxidative Damage via Keap1-Nrf2 Signaling Pathway

Oxidative damage frequently contributes to the development of enteritis diseases. This condition is exacerbated when inflamed intestinal mucosa produces excess ROS, leading to further oxidative damage, sustaining inflammation, and ultimately causing tissue destruction. As shown in Figure 4A, the production of ROS in the Salmonella group was significantly higher than the control group, while SALM + AF40 significantly decreased ROS level. Moreover, Salmonella infection was observed to increase the oxidative damage marker MDA concentration in the colon, whereas AF significantly reduced the MDA concentration (Figure 4B). Furthermore, SALM + AF40 treatment significantly enhanced antioxidative capacity, as demonstrated by elevated activities of CAT (69%), GPx (83%), and SOD (21%) compared to those in the colitis group (Figure 4C). As shown in Figure 4E, Western blotting results also corroborated these findings. The concurrent treatment with Salmonella and AF resulted in a decreased expression of the Keap1 protein. This reduction led to the increased nuclear translocation of Nrf2, thus increasing its nuclear expression levels. Such changes facilitated the expression level of downstream antioxidant enzymes, notably NQO1, HO-1, and SOD2, especially in SALM + AF40 treatment (Figure 4E). Aligning with these observations at the protein level, SALM + AF40 treatment enhanced the expression level of Nrf2, HO-1, and NQO1 mRNA, which were suppressed by Salmonella treatment (Figure 4D). Furthermore, immunofluorescence results showed that the SALM + AF40 treatment enhanced Nrf2 nuclear translocation (Figure 4F), leading to higher levels of HO-1 (Figure 4G) and NQO1 (Figure 4H) protein expression in comparison to the SALM treatment. Collectively, these results suggested that the AF antioxidant characteristics were mediated through the restoration of the Keap1-Nrf2 signaling pathway.

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AF Attenuated Salmonella-Induced Colitis by Maintaining Epithelial and Mucosal Barriers

Alcian blue staining indicated that the positive reaction of acid mucin secreted by goblet cells significantly decreased in Salmonella infection (Figure 5A). To evaluate the role of AF in maintaining the epithelial and mucus barriers, EdU and UEA-1 were detected as markers of cell proliferation and goblet cells, respectively. As shown in Figure 5B,C, Salmonella-induced colitis caused a significant decrease in these markers by immunofluorescence staining, indicating a loss of goblet cells and reduced cell proliferation, which was mitigated by oral administration of AF. Positive cells stained with TUNEL were used to evaluate the colitis-induced cell apoptosis. Colitis induced by Salmonella significantly increased the number of TUNEL-positive stained cells. At the same time, AF significantly reduced cell apoptosis in the mice with Salmonella treatment (Figure 5C). Moreover, the impact of AF on proteins of intestinal epithelial tight junctions and adhesion was evaluated, such as Claudin1, ZO-1, and E-cadherin. Salmonella treatment significantly suppressed the expression of these above proteins, whereas AF countered the Salmonella-induced reduction in their expression levels (Figure 5D). Additionally, Salmonella treatment resulted in a decreased expression level of aquaporin Aqo8 water channel protein, which was restored following AF treatment. Overall, these results reveal that the alleviating effect of AF on Salmonella-induced colitis is also associated with its protection of both mucosal and epithelial barriers.

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AF Modulated Composition of Gut Microbiota and Intestinal Fermentation State

In the assessment of the α-diversity index, we observed that both AF addition and Salmonella infection increased microbial diversity in comparison to the control treatment, as depicted in Figure 6A. Regarding diversity index, the control group showed a lower diversity index than the Salmonella infection group. Principal coordinate analysis (PCoA) further demonstrated that the microbial composition of the AF treatment (SALM + AF40) was similar to that of the control, and it was distinctly different from the Salmonella treatment (Figure 6D). The microbial community composition in the SALM + AF120 treatment resembled that in the Salmonella infection treatment (Figure 6D). At the phylum level (Figure 6B), fecal bacteria composition analysis indicated a significant decrease in the relative abundance of Firmicutes and Verrucomicrobia, whereas the abundance of Bacteroidetes and Proteobacteria increased due to the Salmonella infection. Collectively, these phyla make up over 90% of the bacterial population. Conversely, the relative abundance of Actinobacteria was raised by AF supplementation and the Salmonella infection. At the genus level (Figure 6C), the relative abundance of genus Lactobacillus, Enterorhabdus, RF39, Dubosiella, Faecalibaculum, and Clostridia UCG-014 significantly reduced, whereas the abundance of Butyricimonas, Muribaculum, Bacteroides, Alistipes, and Akkermansia significantly increased in Salmonella treatment. Moreover, the LDA and LEfSe analyses revealed that Dubosiella, Roseburia, Eubacterium ventriosum, and Lachnospiraceae UCG-004 were more abundant in the control (Figure 6E,F). In the SALM + AF40 treatment, the abundance of Akkermansia, Enterorhabdus, and Faecalibaculum was higher than that in the other two groups (Figure 6E,F).

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Furthermore, we utilized Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) to predict the microbial gene functions of the differential microorganisms (Figure 6G). Compared to the control group, SALM treatment significantly upregulated KO pathways such as autophagy, LPS biosynthesis, peroxisome, sulfur metabolism, bacterial invasion of epithelial cells, and protein digestion and absorption. The bacterial secretion system was also enriched, though the SALM treatment was only numerically higher than the control treatment. AF supplementation, especially the SALM + AF40 treatment, significantly reduced the aforementioned KO pathways compared to the SALM treatment. Notably, the flavonoid and isoflavonoid biosynthesis pathways were significantly enriched, although no significant differences were discovered.

To further investigate the impact of these microbial community alterations, we evaluated the concentrations of SCFAs in feces. It was observed that the concentrations of acetate, butyrate, and total SCFAs were significantly reduced by Salmonella-induced colitis (Figure 6H). It is worth noting that AF supplementation could reverse the depletion of these SCFAs. The concentrations of valerate, isobutyrate, and isovalerate showed no significant differences across the different treatments. Taken together, the changes in the intestinal microbial structure alter the intestinal fermentation state by enhancing LPS biosynthesis, oxidative phosphorylation, and bacterial invasion of epithelial cells, thereby resulting in the occurrence of colon inflammation. The reversal of this process through AF intervention may be an important microbial mechanism by which AF alleviates colitis.

Discussion

In this study, the Salmonella-induced colitis mouse model was employed to evaluate the potential therapeutic effects of AF on colitis. The successful establishment of the model was demonstrated by the clinical observations (e.g., diarrhea, body weight loss, and severe colonic inflammation), immune response characterization (e.g., increased pro-inflammation cytokine), gut barrier integrity (e.g., loss of goblet cells and mucus secretion), and gut microbiota composition (e.g., increased abundance of genera Escherichia-Shigella, Oscillibacter, and Erysipelatoclostridium). Our study highlights the multifaceted tissue-protective effect of AF on the prevention or alleviation of colitis in the Salmonella-induced mouse model.

This study showed for the first time that AF can alleviate colitis caused by Salmonella infection through the TLR4/IκB/NF-κB/COX-2 pathway (Figure 3). Consistent with prior research, LPS and endotoxins present in the Salmonella cell wall activate TLR4 and its ligand MyD88, initiating the phosphorylation of downstream protein IκB (C. Y. Chen et al. 2018; Domínguez-Medina et al. 2020). Under normal conditions, NF-κB is inactivated by binding to IκB (Zhao et al. 2021). However, when IκB is phosphorylated, it undergoes ubiquitination and degradation, which releases NF-κB. The phosphorylated NF-κB then translocates to the nucleus, where it interacts with the promoters of various pro-inflammatory genes, such as iNOS, COX-2, and TNFα, thereby initiating gene transcription and aggravating the inflammatory response (Cinelli et al. 2020). AF alleviated this inflammatory process by reversing the cascade described above. Notably, this study also found that AF inhibited the upregulation of the NLRP3 inflammasomes induced by Salmonella (Figure 3D). In line with the previous finding, enhanced NF-κB signaling can enhance the transcription of inflammasome components, including caspase-1, pro-IL-1β, and NLRP3 (He et al. 2021). Then, the oligomerized NLRP3 recruits the adaptor protein ASC, which aggregates pro-caspase-1 and triggers the formation of active caspase-1, completing the assembly and activation of the inflammasome (Swanson et al. 2019). Activated NLRP3 inflammasomes exacerbate the inflammatory response by further promoting the expression of pro-inflammatory cytokines. In summary, AF reduced colitis symptoms in this mouse model by inhibiting the TLR4/IκB/NF-κB/COX-2 pathway and NLRP3 inflammasomes activation.

Oxidative stress occurs when there is an excess of oxidants over antioxidants, resulting in the interruption of redox signaling that ultimately leads to molecular oxidative damage. Previous studies have shown that an imbalance between oxidants and antioxidants is associated with the pathogenesis and progression of colitis (Elmaksoud et al. 2021; Hwang et al. 2020). Under physiological conditions, ROS plays a crucial role in mucosal defense through posttranslational modification. However, excessive ROS leads to nonspecific interactions with biomolecules such as proteins, lipids, nucleic acids, and carbohydrates, resulting in potentially toxic byproducts and destructive oxidative stress (Aviello and Knaus 2017). The overproduction of ROS and MDA, along with an uptick in TUNEL-positive apoptotic cells (Figures 4A,B and 5C), underscored oxidative stress as a key pathophysiological factor in Salmonella-induced colitis in mice. Nevertheless, AF treatment significantly enhanced the clearance of Salmonella-induced ROS, consistent with the in vitro antioxidant results of AF's extensive ROS scavenging capacity (Figure 1B–D). Moreover, a significant increase of antioxidant enzymes (i.e., CAT, SOD, and GPx) in SALM + AF40 and SALM + AF120 groups suggested that AF improved ROS detoxification and reduced oxidative stress (Figure 4C). These results emphasize the effectiveness of AF treatment in mitigating oxidative stress and the resulting damage, thereby helping to attenuate colonic inflammation caused by Salmonella.

To further understand its mechanism of mitigating oxidative stress, this study investigated the Keap1-Nrf2 pathway. Keap1 is a molecular sensor for ROS and other reactive species, monitoring cellular redox status and transmitting signals to Nrf2 (Bellezza et al. 2018). Under normal conditions, Nrf2 undergoes rapid degradation by Keap1. Upon oxidative stress, the DLG motif in Nrf2 is released from Keap1's DGR domain, preventing its ubiquitination and degradation, thus enabling Nrf2's nuclear translocation and further activating various antioxidant and detoxification genes (Liu et al. 2021). This study observed that Salmonella disrupted the Keap1-Nrf2 signaling axis by increasing Keap1 levels and inhibiting Nrf2 (Figure 4E,F). Conversely, AF treatment restored Nrf2's function, effectively reversing Salmonella’s suppressive impact on the target antioxidant genes such as HO-1 and NQO-1 (Figure 4E,G,H). Intriguingly, crosstalk between the Keap1-Nrf2 and NF-κB pathways seems to attenuate oxidative damage synergistically in this model. Previous studies have reported that Rac1 mediated NF-κB's induction of Nrf2, escalating HO-1 expression, which in turn reduced NF-κB's inflammatory signaling (Ranneh et al. 2019; Zhao et al. 2021). In this research, AF effectively counteracted the suppressive impact of Salmonella on Nrf2 nuclear translocation and antioxidant gene expression, ultimately preventing inflammatory responses mediated by NF-κB. This demonstrated that Nrf2 may play a critical role in alleviating inflammation driven by NF-κB activation. Taken together, AF inhibited oxidative stress induced by Salmonella via the reconstituted Keap1-Nrf2 pathway.

Furthermore, AF mitigated Salmonella-induced intestinal barrier damage in the colon (Figure 5). Goblet cells and their secreted mucin are essential for maintaining the intestinal mucus barrier (Chami et al. 2020). The intestinal mucus layer safeguards epithelial cells by maintaining a spatial separation between the colonization of symbiotic bacteria and the host's epithelial barrier. Dysfunctions in goblet cells, such as reductions in their numbers and adverse changes in mucin synthesis, are linked to IBD (Bastaki et al. 2016). The loss of goblet cells and the mucin may enable Salmonella and other pathogens to directly contact epithelial cells, thereby leading to the onset and progression of colitis. A previous study has reported that the release of TNF-α during intestinal inflammation can disrupt mucin production by goblet cells (Ren et al. 2018). Similarly, a significant increase in TNF-α was observed in the Salmonella group. As expected, AF enhanced intestinal epithelial homeostasis by reconstructing the mucosal barrier and isolating pathogens from epithelial cells. This effect was supported by the observed upregulation of tight junction proteins and the mucin marker UEA-1. In summary, AF effectively mitigated disruptions in intestinal barriers and epithelial functions caused by Salmonella-induced colitis, suggesting potential protective mechanisms that merit clinical investigation.

The mitigation of intestinal barrier disruptions by AF may be attributed to changes in the composition of the gut microbiota and the state of intestinal fermentation. In the Salmonella infection group, an increased α-diversity of intestinal bacteria was observed (Figure 6A), consistent with findings in other Salmonella mouse experiments (He et al. 2021; Yuan et al. 2022). The increased α-diversity of intestinal bacteria might reflect dysfunction in the intestinal microbiota structure caused by Salmonella infection. It is worth mentioning that a reduction in Firmicutes is associated with elevated intestinal inflammation, whereas a decrease in Bacteroidetes is typically correlated with diminished intestinal inflammation (Stojanov et al. 2020). In the present research, similar patterns were detected, and Salmonella infection has significantly increased relative abundance of Bacteroidetes (Figure 6B). Furthermore, supplementation with AF enhanced the intestinal microbiota's functionality and mitigated Salmonella-induced colitis by increasing the abundance of beneficial bacterial genera, specifically Dubosiella and Akkermansia (Bourgonje et al. 2023). The role of Dubosiella in reducing oxidative stress, boosting endothelial function, and modulating the intestinal microbiota has been reported in a previous study (Liu, Wang, et al. 2023). Akkermansia muciniphila, as a member of the Verrucomicrobia phylum, plays a crucial role in maintaining intestinal barrier integrity by modulating the host's immune response and reducing local inflammation (Derrien et al. 2017; Herfindal et al. 2022; Y. Wang et al. 2024). In this study, AF suppressed the increase in bacterial genera, such as Escherichia-Shigella, Oscillibacter, and Erysipelatoclostridium, in Salmonella-induced mice (Figure 6E). Escherichia coli-Shigella, which belongs to the Proteobacteria phylum, is involved in intestinal inflammation by penetrating epithelial barriers, inducing apoptosis in infected macrophages, and releasing IL-1β (Allaire et al. 2018). The potential colonic disease pathogen Erysipelatoclostridium has been documented to proliferate in inflammatory bowel disease and exhibited a positive association with TNF-α levels (Cheng et al. 2023; Gowd et al. 2019; Yu et al. 2023), which is in line with the outcomes of our study. Oscillibacter is related to inflammatory bowel disease, with its abundance increasing after Salmonella treatment. Oscillibacter can enhance intestinal permeability by upregulating the inflammatory cytokine IL-6 (Wu et al. 2019). Additionally, Oscillibacter was enriched in a colorectal cancer mouse model (Wang et al. 2018. In the current experiment, the SALM + AF40 treatment significantly increased the relative abundance of butyrate-producing bacteria, such as Clostridia-UCG-014, Eubacterium, Roseburia, Faecalibacterium, and Ruminococcus, which was accompanied by a higher butyrate concentration (Figure 6E,H). Intestinal butyrate-producing bacteria are predominantly responsible for butyrate synthesis through carbohydrate fermentation and serve as the primary source of butyrate in the intestine. Previous studies have reported that butyrate supplies approximately 60%–70% of the energy required by colon epithelial cells and serves as the principal energy supplier for the intestinal mucosa (Liu et al. 2018). Butyrate not only provides energy and promotes the repair of the intestinal mucosal barrier but also exhibits anti-inflammatory properties by inhibiting inflammatory mediators. Additionally, butyrate-producing bacteria are instrumental in maintaining intestinal barrier integrity by restraining the NLRP1 inflammasome activity and curtailing the release of pro-inflammatory cytokines (Tye et al. 2018). Similar to butyrate, the concentration of acetate and total SCFAs also significantly increased in the AF group in the current experiment (Figure 6H). SCFAs are critical physiological messengers that bridge the symbiotic microbiota with the mucosal immune defense (Meng et al. 2024). SCFAs induce the activation of innate lymphocytes and regulatory T cells, which are indispensable for the maintenance of intestinal mucosal immunity (Li et al. 2020; Yu et al. 2023). Additionally, it is known that SCFAs suppress inflammatory factor expression by promoting histone acetylation, activating GPRs, or inhibiting the NF-κB signaling cascade (Gowd et al. 2019). A recent study has reported that SCFAs decreased in IBD patients’ feces (Liu et al. 2023). Furthermore, it has been reported that SCFAs can enhance tight junction proteins, thereby maintaining the intestinal barrier (Saleri et al. 2022). AF was found to be involved in upregulating the expression levels of tight junction and adhesion proteins that were diminished by Salmonella (Figure 5D). Therefore, it is inferred that AF alleviated intestinal inflammation and barrier impairment caused by Salmonella in this model partly by optimizing the gut microbial community composition and regulating the production of SCFAs.

Although our study provides valuable insights into the protective effects of AF in the context of Salmonella-induced colitis, several limitations warrant further investigation. First, the long-term effects of AF supplementation on chronic colitis and the prevention of inflammatory relapses remain unexplored. Future research is warranted to evaluate AF's sustained impact on chronic colitis models and its potential role in mitigating recurrent inflammation. Second, this study assessed two doses of AF (40 and 120 mg/kg), with the higher dose (120 mg/kg) yielding suboptimal results. Future research is necessary to conduct comprehensive dose–response analyses based on findings of this study to optimize the protective potential of AF. Third, although we observed modulation of the intestinal microbiota, the precise molecular mechanisms underlying AF's regulatory effects on the microbiota remain unclear. Additional research is required to clarify the interaction between AF and gut microbiota, as well as its metabolites at the molecular level, which may enhance understanding of its potential protective effects on gut health. Further elucidation of pertinent mechanisms needs to be conducted, including the employment of molecular docking techniques. Finally, although the Salmonella-induced colitis model is widely utilized, the results may not fully extend to colitis caused by other pathogens or to genetically predisposed individuals. Additional studies using diverse models are essential to confirm the broader applicability of AF's protective effects across different types of colitis.

Conclusion

In conclusion, AF showed excellent antioxidant activity against ROS and its derivatives in vitro. In vivo, AF effectively alleviated colitis symptoms induced by Salmonella and directly scavenged the ROS. AF also increased antioxidant activity through the activation of the Keap1-Nrf2 antioxidant pathway and suppressed the expression of pro-inflammatory cytokines by inhibiting the activities of IκB/NF-κB phosphorylation-mediated TLR4/NF-κB/COX-2 signaling pathway. Furthermore, AF maintained epithelial barrier homeostasis through the upregulation of tight junction proteins and UEA-1, which was attributed to an alteration in the composition of the gut microbiota and intestinal fermentation state. In summary, AF exerted multifaced protective effects against Salmonella-induced colitis by weakening oxidative stress, maintaining intestinal barrier integrity and microbial homeostasis, thereby attenuating inflammation. Besides providing symptomatic relief for colitis, the study results also indicate the preventive impact of AF on inflammation. These findings highlight AF's potential application value as a promising phytopharmaceutical for both the prophylaxis and treatment of colitis and related diseases caused by oxidative stress.

Author Contributions

Huawei Su and Yang He designed the study. Xiaoli Qin and Yan Lu performed the experiments. Xiaoli Qin analyzed the data and wrote the initial manuscript. Yafang Cui, Yawen Luo, Shengnan Min, and Wenfang Wang helped with the experimental section. The manuscript was edited by Kai Zhao, Muhammad Aziz ur Rahman, and Fuyu Yang. Huawei Su, Binghai Cao and Yang He reviewed and revised the manuscript.

Acknowledgments

We are deepest grateful to the editor and the anonymous reviewers for their careful work and thoughtful comment, which greatly enhanced the quality of this article.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data will be made available on request.

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