2.1 Material and chemicals

Methanol (CH3OH) (GR, ≥ 99%), acetonitrile (ACN) (GR, ≥ 99%) were purchased from Merk (German). Formylic acid (HCOOH) (GR, ≥ 98%), naringin (98.75%), phloridzin (98.55%) and dextran sulfate sodium (DSS) (AR) were purchased from Aladdin (Shanghai, China). L-2-phenylalanine (AR) was purchased from Brylwilliam (China). Sodium chloride injection (0.9%) was purchased from Wuhan Binhu Shuanghe Pharmaceutical Company. acetate, propionate, and butyrate, isobutyrate, n-valerate, isovalerate, n-caproate, and the internal standard 2,2-dimethylbutyrate were gas chromatographic standards purchased from Aladdin.

2.2 Animals and design

A total of 54 male C57BL/6 mice (6–8 weeks old; 21–23 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China), acclimatized for one week, and then randomly assigned to four groups: a Control group (n = 10), a DSS group (n = 20), a Naringin group (n = 12), and a Phloridzin group (n = 12). As outlined in the experimental design (Fig. 1A), ulcerative colitis was induced over seven days in the DSS, Naringin group and Phloridzin group by administering 2% (w/v) DSS in the drinking water, which was replaced every 48 h. Throughout this induction period, mice in the Naringin and Phloridzin groups received daily oral gavages of naringin (100 mg/kg) and phloridzin (100 mg/kg), respectively, while the Control group and DSS group received an equivalent volume of saline. On the morning of day 8, faecal samples were collected and stored at -80°C, after which the mice were dissected and colon tissues were harvested for subsequent analysis. All animal experiments were approved by the Committee on Care and Use of Laboratory Animals of South Central Minzu University, China (Approval No. 2022-scuec-38).

Fig. 1

Experimental design of the DSS-induced UC mouse model and effects of naringin and phloridzin on UC mice. A Experimental design. B Body weight change measurement. C Colon length measurement(*, p < 0.05; ****, p < 0.0001, compared with the control group). D Macroscopic morphology of the colon. E DAI index. F Histological evaluation (H&E staining 100 ×)

2.3 Histopathological assessment

Harvested colon tissues were fixed in 10% neutral-buffered formalin, processed through a graded ethanol series for dehydration, and embedded in paraffin. The paraffin blocks were then sectioned and stained with haematoxylin and eosin (H&E) for histological evaluation.

2.4 Histological immunochemistry and inflammatory factor assay

Quantitative detection of TNF-α, IL-17 and IL-22 levels in mucosal tissue and serum using commercial ELISA kits. Tissue samples were rinsed with PBS and homogenized in tissue protein extraction reagent. The homogenates were repeatedly shaken and pipetted, followed by centrifugation to collect the supernatant. Protein concentrations were determined using a BCA Protein Assay Kit. After sample preparation and antibody incubation, optical density values of the target protein bands were analysed with AlphaEaseFC software.

2.5 Non-targeted metabolomics analysis

Briefly, the cecum content (40 mg) was extracted with the internal standard extract (containing 70% methanol). The supernatant was centrifuged three times at 12,000 rpm for 10 min, under freezing conditions and filtered through 0.22 μm polyvinylidene difluoride (PVDF) membrane for further analysis.

Non-targeted metabolomics employs ultra-performance liquid chromatography (UPLC) (ExionLC AD, https://sciex.com.cn/) and quadrupole-time-of-flight mass spectrometry (TripleTOF 6600, AB SCIEX). Column: Waters ACQUITY UPLC HSS T3 C18 1.8 µm, 2.1 mm × 100 mm. Flow rate: 0.35 ml/min; Column temperature: 40 °C; Injection volume: 5 µl. Positive: Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. Negative: Mobile phase A was ultrapure water and mobile phase B was acetonitrile; Gradient conditions: 0–11 min, 5–90% B; 11–12 min, 90% B; 12–12.1 min, 90–5% B; 12.2–14 min, 5% B. Specific mass spectrometry conditions are detailed in the supporting information. Specific mass spectrometry conditions are detailed in the supporting information (Table S1).

2.6 Widely targeted metabolomics analysis

The widely targeted metabolomics assay was performed by Metware (Wuhan, China). Widely-targeted metabolomics employs ultra-performance liquid chromatography (UPLC) (ExionLC AD, https://sciex.com.cn/) and tandem mass spectrometry (MS/MS) (QTRAP®, https://sciex.com/). Metabolite quantification was completed by analysis in MRM mode based on Q-Trap triple quadrupole mass spectrometry. Column: Waters ACQUITY UPLC HSS T3 C18 1.8 µm, 2.1 mm × 100 mm. Flow rate: 0.35 ml/min; Column temperature: 40 °C; Injection volume: 2 µl; Positive: Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. Negative: Mobile phase A was ultrapure water and mobile phase B was acetonitrile; Gradient conditions: 0–11 min, 5%-90% B; 11–12 min, 90% B; 12–12.1 min, 90%-5% B; 12.2–14 min, 5% B. Mass spectrometry conditions same as in Sect. 2.5.

2.7 Data analysis

Mass spectrometry data using public databases (including Metlin, HMDB, KEGG databases) and MetDNA, to obtain the multi-ion pair information and retention time of the identified metabolites. The most plausible batch of compounds was expanded for data analysis of the subsequent broad-target mass spectrometry assay. Metabolite quantification was completed by analysis in multiple reaction monitoring (MRM) mode based on Q-Trap triple quadrupole mass spectrometry. Data acquisition was performed using Analyst 1.6.3, and integration (quantification) was performed using MultiQuant 3.0.3 after down-conversion. Normalized data were subjected to Principal Component Analysis (PCA) and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) in R. Differential metabolites were screened based on the variable importance projection (VIP) of the OPLS-DA model in the multivariate statistical analysis, combined with the P-value and difference multiplicity value (FC) of the t-test of the univariate analysis, and the following criteria were used for the screening: VIP ≥ 1, FC ≥ 2 or FC ≤ 0.5, P-value ≤ 0.05. Pathway enrichment analysis was performed using the KEGG database. The diagnostic accuracy of potential biomarkers was assessed using subject working curves (ROC curves), and the relationship between DAI and potential biomarkers was analysed by linear regression.

2.8 Targeted metabolomics analysis of tryptophan

20 mg of faecal sample mixed with 400 μL of internal standard extraction solution (70% methanol in water) and vortexed for 6 min, followed by sonication for 10 min. Next, the sample was vortexed again for 3 min and incubated at -20 °C for 60 min. After centrifuged at 12,000 rpm for 10 min, 200 μL of supernatant was injected into the analytical instrument for subsequent measurement and analysis.

The LC-QqQ-MS was used for the targeted detection of tryptophan pathway metabolites with an injection volume of 1 μL, column temperature of 40 °C, mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid in acetonitrile at a flow rate of 0.4 mL/min, and the chromatographic column was a Waters ACQUITY UPLC C18 + (2.1 × 50 mm, 1.7 µm). Gradient conditions: 0–2 min, 3–50% B; 2–2.5 min, 50–60% B; 2.5–3 min, 60–97% B; 3–3.5 min, 97–3% B; 3.5–5 min, 3% B. Detection was performed by negative multiple reaction monitoring (MRM). The nitrogen dry gas temperature was 300 °C and the nitrogen sheath gas temperature was 250 °C. The nitrogen dry gas flow rate was 5 L/min and the nitrogen sheath gas flow rate was 11 L/min. The capillary voltage was 3500 V, the nebulizer gas pressure was 45 psi, and the nozzle voltage was 500 V. The nozzle voltage was 500 V. The nozzles were designed to be used in a variety of applications. Specific MRM information is shown in Table S5.

2.9 Targeted metabolomics analysis of SCFA

A total of 25 mg samples were mixed with 10 μL of internal standard (1 mM, 2,2-dimethylbutyric acid), 400 μL of 1 M HCl and then add 400 μL of EE after three freeze–thaw cycles. 200 μL of centrifugal separation supernatant (4 °C, 12,000 rpm, 10 min) was injected into the sample to be measured and analysed.

The 6890N-5973N Gas Chromatograph-Mass Spectrometer and a HP-INNOWAX (60 × 0.250 × 0.25 μm) gas chromatographic column was used, with helium as the carrier gas at a flow rate of 0.8 mL/min, and the inlet temperature was 250 °C, the ion source temperature was 230 °C, the capillary temperature was 250 °C, and the total injection time was 29 min: the temperature was 100 °C for 2 min; the temperature was increased to 120 °C and kept for 3 min, the temperature increase rate was 10 °C/min; the temperature was increased to 150 °C and kept for 6 min, the temperature increase rate was 510 °C/min; the temperature was increased to 250 °C and kept for 6 min, the temperature increase rate was 25 °C/min. Quantification was performed based on the relative peak area using acetate and 2, 2-dimethylbutyric acid as the internal standard.