# Helile formula from “Tai ping sheng hui fang”: anti-Helicobacter pylori activity, gut microbiota modulation, and inflammatory response regulation

## TL;DR

This study explores the Helile formula's ability to fight Helicobacter pylori, reduce gut inflammation, and alter gut bacteria, offering a potential alternative treatment.

## Contribution

The study identifies specific compounds in Helile formula and demonstrates its in vitro and in vivo anti-Helicobacter pylori effects and gut microbiota modulation.

## Key findings

- Helile formula disrupts Helicobacter pylori structure and inhibits toxin-related genes.
- The formula modulates gut microbiota, particularly Muribaculaceae and Lactobacillaceae families.
- It reduces inflammation and cell adhesion in mice infected with Helicobacter pylori.

## Abstract

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. 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 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.

Flowchart detailing the composition and analysis of the Helile Formula, originating from the “Taiping Shenghuifang”. The formula includes Sanguisorba officinalis L. and Terminalia chebula Retz. in a 1:2 ratio. Research involves in vitro methods like MIC assay, SEM, RT-QPCR, and in vivo methods such as Western blot and Metabolomics Analysis.