Portulaca oleracea L. plants were purchased from Huangshan, Anhui Province, China. Ten grams of the dried plant materials was smashed equably and soaked in 100 mL of 50% ethanol at 25°C in a sealed beaker for 24 h in the dark. Then, the soaked solution was collected, and the powder was extracted twice with a fresh batch of 100 mL of 50% ethanol for 2 h by ultrasonic treatment at 100 W. Thereafter, the two extracts were mixed, filtered by suction, and concentrated into a thick extract without excess water and ethanol using a rotary evaporator at a low speed and 70°C. High-performance liquid chromatography (HPLC) grade ethanol was purchased from RCI Labscan Limited, Pathumwan, Bangkok, Thailand), and Milli-Q water was prepared using a Milli-Q purification system (Millipore, MA, United States). The concentrated extract was stored overnight at -80°C, followed by freeze-drying with a VirTis freeze dryer (VirTis Company, New York, United States) for 2 d to obtain the freeze-dried powder extract of purslane.

The purslane extract was identified using ultra performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS/MS; Waters, MA, United States). The liquid chromatographic separations were performed on a Waters Acquity UPLC H-Class system (Waters, MA, United States) equipped with a Waters Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm), with the column temperature set to 30°C. The mobile phase consisted of 0.1% formic acid in water (phase A) and acetonitrile (phase B). HPLC grade acetonitrile was purchased from Merck (Darmstadt, Germany). The flow rate was set to 0.3 mL/min, and the injection volume was 1 µL. The samples were eluted with a linear gradient: 5%B for 0–1 min; 5%–95%B for 1–28 min; 95%–5%B for 28–36 min; 5%B for 36–40 min.

The MS analysis was performed on a Waters Xevo G2-XS QTOF system combined with an electrospray ionization (ESI) source (Waters, MA, United States). The mean-squared error centroid mode was used for sensitivity and negative ESI mode was used to collect fragment ions within the m/z range of 10–1,500 with the scan time set to 1 s. The tuning parameters were set to a capillary voltage of 2 kV, sampling cone voltage of 40 V, source offset of 80 V, source temperature of 120°C, desolvation temperature of 450°C, cone gas flow rate of 50 L/h, and desolvation gas flow rate of 700 L/h. The data were processed using MassLynx V 4.1 software (Waters).

The intestinal tissues were obtained from all groups of mice, divided into large and small intestine parts, frozen with liquid nitrogen, and stored at −80°C. Approximately 1.5-cm-length tissues samples from the same part of the colon were homogenized using saline and precooled instruments. The supernatant was collected after the homogenate was centrifuged for 10 min at 2,500 rpm/min and 4°C. The IL-6 and TNF-α concentrations were determined in all mice intestinal tissues using ELISA kits following the manufacturer’s instructions (NeoBioscience, Shenzhen, China).

Protein concentrations in the colon tissue lysates were measured via the bicinchoninic acid (BCA) assay (ThermoFisher Scientific, MA United States). Equal amounts of samples were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corp., MA, United States) on ice by the wet transfer mode (BIO-RAD, United States). After blocking with 5% defatted milk and tris-buffered saline (TBS) containing 0.1% Tween-20, the membrane was probed with appropriate primary antibodies diluted to 1:1,000 against TLR4, MyD88, phosphorylated NF-κB inhibitor alpha (IκB⍺) at Ser32, total IκB⍺, phosphorylated p65 at Ser536, total p65, and β-actin (Cell Signaling Technology, United States) overnight at 4°C. The protein bands were then scanned using the ChemiDocTM MP imaging system (BIO-RAD, United States) after incubation with corresponding secondary antibodies (Cell Signaling Technology, United States) using the Supersignal TM West Femto maximum sensitivity substrate (ThermoFisher Scientific, PA, United States). The band intensities were quantified using Image Lab software (Version 3.0).

Small intestine samples from all mice from roughly the same locations were collected and fixed in 4% paraformaldehyde solution. Then, the samples were embedded in paraffin and sliced into 5-μm-thick sections before being stained with hematoxylin and eosin (H&E; Beyotime Biotechnology, Shanghai, China). Additionally, immunohistochemistry (IHC) analyses of IL-6, TNF-α, TLR4, and phosphorated p65 (Ser536) were conducted as described by Liu et al. (2019). The paraffin sections were dewaxed, and the antigens were retrieved with EDTA (pH 9.0) and gradient organic solvents, followed by 2-h of blocking with 5% bovine serum albumin (BSA) and TBS with Tween-20 as well as blocking with 3% H₂O₂ (aq) for 25 min at room temperature. Next, the sections were incubated with the primary antibodies of IL-6, TNF-α, TLR4, and phosphorated p65 at Ser536 (1:100, Cell Signaling Technology, United States) overnight at 4°C, followed by incubation with the corresponding secondary antibodies. The staining was determined using an I-View DAB detection system (Servicebio, Beijing, China), and the sections were stained with hematoxylin (Beyotime Biotechnology, Shanghai, China) to visualize the nuclei before being dehydrated. All the stained specimens were observed and recorded using an Olympus IX73 microscope. The H&E sections were photographed at ×40 and ×200 magnifications, and the IHC sections were photographed at ×400.

The data were recorded as mean ± standard error of the mean (SEM) for all experiments. For the statistical analysis, GraphPad Prism (Version 9.1.1, United States) was used to obtain multiple comparisons between the experimental groups by one-way ANOVA and unpaired Student’s t-test. A p-value <0.05 was deemed to be significant.

Eight amide components were identified from the prepared purslane extract, which are shown in the extracted ion chromatogram (EIC) in Figure 1. The information on the identification of the eight components in the purslane extract by ESI-QTOF-MS/MS in the negative ESI mode is shown in Table 1, including the retention time, observed mass, theoretical mass, predicted chemical formula, and observed MS/MS fragment ions. The identification results and bases are also listed in Table 1. Here, N-trans-feruloyltyramine matched with the MS/MS result in the PubChem database; oleracein A, B, C, D, and E along with N-caffeoyldopamine were identified by referring to the MS/MS information in literature (Jiao et al., 2014); lastly, N-cis-feruloyltyramine was identified only using the exact mass in this study.

The DIO mice were administered purslane extracts at 200 mg/kg/day by oral gavage for a total duration of 4 weeks to investigate its anti-inflammatory effects on intestinal tissues in vivo. Before commencing the experiments, the mice had similar bodyweights and were randomly assigned into different groups. As shown in Figure 2A, after the 14-week HFD induction, the final bodyweights of the DIO mice were much higher than those of the control mice that received a standard chow diet, indicating successful establishment of obese mouse model. Interestingly, the 4-week oral administration of purslane extract seemed to produce a slight but non-significant weight-reducing effect on the DIO mice. Figure 2B shows that the colon length of a representative DIO mouse was obviously shorter (5.5 cm) than that of the control mouse (7.2 cm), which was significantly improved by chronic purslane treatment (6.4 cm). The summarized graph of the colon lengths of all groups of mice is shown in Figure 2C, indicating that purslane extract alleviates colon shortening in DIO mice.

To investigate the anti-inflammatory capabilities of purslane extract, the expression levels of pro-inflammatory cytokines IL-6 and TNF-α in the colon tissues were determined using ELISA kits. The IL-6 and TNF-α levels were upregulated sharply in the DIO mice but reversed significantly after purslane treatment to levels comparable to those of the control group (Figures 3A, B). These results illustrate that a 4-week oral administration of purslane extract (200 mg/kg/day) could suppress intestinal inflammation in DIO mice.

Colon tissues of all groups of C57BL/6J mice were obtained to further study the underlying action mechanisms of purslane by Western blotting. It was discovered that compared to the control group, the TLR4, MyD88, Ser32 phosphorylation of NF-κB inhibitor alpha (IκBα), and Ser536 phosphorylation of NF-κB p65 expression levels (compared to β-actin or related total proteins) were all remarkably upregulated in the colons of DIO mice (Figures 4A–D). Purslane extract treatment significantly reduced the protein expressions of TLR4 and MyD88 as well as the phosphorylation of IκBα and NF-κB p65 in the colon sections, suggesting inactivation of the TLR4/MyD88/NF-κB signaling pathway and contributing to inflammation relief.

H&E staining was used to observe the histopathological changes in the mouse small intestine tissue, and the corresponding experimental results are shown in Figure 5. The intestinal mucosal structures of the mice in the control group had normal morphologies, with shallow crypts, neatly arranged intestinal villi, and obvious boundaries. In the DIO group, the intestinal villi were disordered with missing top structures, deep crypts, and indistinct boundaries, and a part of the intestinal cortex was separated from the lamina propria. Nevertheless, 4-week oral administration of purslane extract was observed to significantly improve the intestinal injury status and prevent reduction of the ratio of intestinal villus length to crypt depth (p <0.01) in the DIO mice (Figures 5A,B); this ratio of villus length to crypt depth is an important index for evaluating the structural integrity of the small intestinal mucosa. To conclude, chronic treatment with purslane extract was found to improve bowel histopathological injury in DIO mice effectively.

In line with the results of the inflammatory marker measurements in the mouse colons, IHC analysis of the small intestine tissues illustrated elevated expression levels (indicated by brown color) of IL-6, TNF-α, TLR4, and phosphorylated NF-κB p65 (Ser536) in the DIO mice and that such elevation was lowered after oral treatment with purslane extract (Figure 6). This phenomenon demonstrates that purslane extract ameliorates intestinal inflammation by suppressing the TLR4/NF-κB pathway in the small intestine tissues of DIO mice.