Helile Formula from "Tai ping sheng hui fang": Anti-Helicobacter pylori Activity, Gut Microbiota Modulation, and Inflammatory Response Regulation

Ling Ou¹Meiyuun Chen¹Chang Peng²Haobo Chen¹Yajie Hao³Qingchang Chen⁴Zhong Feng^1,3*Meicun Yao^1*Xianhe Kong^5,6*

• ¹Sun Yat-sen University, Guangzhou, China
• ²Discipline of Chinese and Western Integrative Medicine，Jiangxi University of Chinese Medicine，Nanchang 330004, China, Nanchang, China
• ³International Pharmaceutical Engineering Lab of Shandong Province, Feixian 273400, Shandong, China., Feixian, China
• ⁴Department of Biomedical Engineering, College of Design and Engineering, National University of Singapore, Singapore, Singapore
• ⁵Department of Gastrointestinal Endoscopy, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
• ⁶Clinical Medical Research Center for Plateau Gastroenterological Disease of Xizang Autonomous Region, Affiliated Hospital of Xizang Minzu University, Xianyang, China

Helicobacter pylori (HP) is a major gastric pathogen linked to chronic gastritis, peptic ulcers, and gastric cancer. The emergence of antibiotic resistance has prompted the search for alternative treatments. The Helile formula, derived from the ancient "Taiping Shenghuifang," is known to treat various degrees of diarrhea and has potential for treating gastrointestinal disorders. However, the antibacterial efficacy, material basis, and action mechanisms of the helile formula against HP remain undetermined.The chemical constituents were analyzed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and high-performance liquid chromatography (HPLC). The in vitro anti-HP activity and underlying mechanisms were investigated through a series of assays, including the determination of minimum inhibitory concentration (MIC), N-phenyl-1-naphthylamine (NPN) uptake assay, scanning electron microscopy (SEM) to observe morphological changes, cell viability and cell adhesion activity assays, assessment of nitric oxide (NO) production, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for gene expression analysis. For the in vivo anti-HP infection study and mechanism exploration, techniques such as hematoxylin and eosin (H&E) staining for histological examination, enzyme-linked immunosorbent assay (ELISA) for cytokine and antibody quantification, 16S ribosomal DNA (16S rDNA) sequencing for 3 microbial community profiling, and metabolomics for global metabolite analysis were employed.Multiple constituents of helile formula, namely ellagic acid, gallic acid, chebulagic acid, chebulic acid, and corilagin, were identified. In vitro, helile formula increased bacterial outer-membrane permeability, disrupted HP structure, inhibited toxin-related genes, and suppressed cell adhesion. In male Kunming mice, helile formula effectively reversed HP-induced inflammation. It modulated key metabolites, such as adenine, panaxytriol, 4-hydroxyglutamate semialdehyde, and N-alpha-Acetyl-L-lysine.It influenced the gut microbiota, especially families like Muribaculaceae and Lactobacillaceae. Adenine, in particular, repaired HP-caused damage to GES1 cells, reduced HP -mediated cell adhesion, and inhibited HP-induced interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) production.These findings demonstrated the remarkable anti-HP efficacy of the helile formula in vitro and in vivo, suggesting that the helile formula represents a highly promising therapeutic candidate for the management of HP infections.