Shouhui Tongbian capsule (SHTB), a commercial Chinese patent medicine, has been shown to modulate gut microbiota dysbiosis and contains antibacterial active ingredients against Helicobacter pylori (H. pylori). H. pylori is a Gram-negative bacterium that colonizes the gastric mucosal and disrupt gastrointestinal microbiota balances. However, the precise mechanism underlying the anti-H. pylori effects of SHTB remains unclear. To investigate the in vitro anti-H. pylori effects and underlying mechanisms of SHTB. High-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), and network pharmacology analyses were used to identify the active compounds and potential targets of SHTB associated with H. pylori infection. The anti-H. pylori activity of SHTB was determined using the broth dilution method. Mechanistic studies were conducted through techniques such as electron microscopy observation, immunofluorescence staining, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blotting analyses. A total of 42 compounds were identified in SHTB. Database screening yielded 365 target genes related to SHTB constituents and 1035 genes associated with H. pylori infection. In vitro assays demonstrated that the minimum inhibitory concentration (MIC) of SHTB against both susceptible and resistant H. pylori strains ranged from 160 to 320 μg/mL, with the minimum bactericidal concentration (MBC) being twice the MIC. No antagonistic effects were observed when SHTB was combined with clarithromycin, metronidazole, levofloxacin, or amoxicillin. SHTB disrupted H. pylori morphology, reduced urease activity, and downregulated virulence-associated gene expression. Cellular assay further indicated that SHTB enhanced the anti-adhesion capability of host cells. Furthermore, SHTB alleviated H. pylori infection-induced damage by inhibiting the abnormal activation of the PI3K/AKT, MAPK, and NF-κB signaling pathway. These findings provide a theoretical basis for elucidating the mechanism of SHTB against H. pylori infection and confirm its protective role in combating H. pylori infection through the regulation of the PI3K/AKT, MAPK, and NF-κB signaling pathway network.

Background and Objective: Indigo and indirubin are the main active components of the traditional Chinese medicine Qingdai, known for their heat-clearing, detoxifying, antibacterial, and anti-inflammatory effects. Indirubin has already been applied clinically with proven safety. Both compounds show potential against drug-resistant Helicobacter pylori infection; however, their strong hydrophobicity limits further application. This study aimed to construct food-grade self-assembled nanoparticles using electrostatic and hydrophobic interactions between ovalbumin and fucoidan to efficiently encapsulate indigo and indirubin, thereby improving their solubility and evaluating the in vitro anti- Helicobacter pylori activity and underlying mechanisms of the three formulations. Methods: Nanoparticles were prepared and characterized. The antibacterial activity was assessed using the broth microdilution and checkerboard methods. The mechanisms were further investigated through network pharmacology, molecular docking, electron microscopy, urease activity assay, RT-qPCR, Western blotting, and untargeted metabolomics analyses. Results: At a carrier concentration of 0.75 mg/mL and a drug loading concentration of 40 μg/mL, the nanoparticles exhibited optimal stability, with an encapsulation efficiency of 68.64% and a loading capacity of 3.66%. Indigo, indirubin, and their nanoparticles showed significant inhibitory and bactericidal effects against both sensitive and drug-resistant Helicobacter pylori strains, with minimum inhibitory concentration ranges of 2.5–5, 5–32, and 2.5–5 μg/mL, respectively. The nanoparticles demonstrated superior efficacy and exhibited synergistic or additive effects when combined with antibiotics. The underlying mechanisms involved disruption of bacterial structures; downregulation of virulence genes related to flagella, adhesion, urease, and VacA; inhibition of urease activity and CagA expression; and interference with key metabolic pathways. Conclusion: Encapsulation of indigo and indirubin by the ovalbumin/fucoidan nanoparticle system significantly enhanced their solubility and anti-Helicobacter pylori activity, providing an experimental basis for developing novel natural therapeutics against Helicobacter pylori infection.