UHPLC-Q/Orbitrap HRMS combined with spectrum-effect relationship and network pharmacology to discovery the gastrointestinal motility-promoting material basis in Citri Sarcodactylis Fructus.

Ethnopharmacological Relevance: The prevalence of gastrointestinal motility disorders (GMD) is increasing and is characterized by long-term recurrence. Citri Sarcodactylis Fructus (CSF), as a traditional Chinese medicine (TCM) known in "regulating qi and harmonizing the stomach", has therapeutic effects on GMD. However, the material basis of its efficacy is not clear.

Aim Of The Study: The aim of this study was to evaluate the gastrointestinal motility-promoting activity of CSF extracts and to screen their active ingredients and to perform a preliminary validation.

Methods: The chemical composition spectrum of different extracts of CSF were established by ultra high-performance liquid chromatography coupled with quadrupole orbitrap high-resolution mass spectrometry (UHPLC-Q/Orbitrap HRMS). The gastrointestinal motility-promoting activities of CSF were investigated by determining the intestinal propulsion rate, gastric emptying rate, acetylcholinesterase activity, and motilin content in L-arginine-induced GMD mice. Spectrum-effect relationship and network pharmacology analysis were used for the screening of potential active ingredients. A zebrafish gastrointestinal motility model traced with Nile Red was established to validate the active ingredients. Molecular docking prediction was used to explore the mechanism of action of the active ingredient. Finally, Western blotting and TUNEL staining were performed to validate the molecular docking predictions.

Results: In total, 42 shared components were identified. The main active fraction of CSF to promote gastrointestinal motility was 70% ethanol elution fraction. Eleven potential active ingredients were screened by grey correlation analysis, orthogonal partial least squares analysis, and "active ingredient-target" network. Six compounds were confirmed as the pharmacodynamic substances of CSF by zebrafish gastrointestinal motility model, namely, quercetin, kaempferol, isorhamnetin, diosmetin, hesperetin, and 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone. Molecular docking predictions and Western blotting assays indicated that CSF may act on AKT and MMP9 targets to exert gastrointestinal motility-promoting activity.

Conclusion: This study provided a foundation for elucidating the gastrointestinal motility-promoting activity of CSF and its material basis by integrating spectrum-effect relationship and network pharmacology. It also provided a theoretical basis for quality control of CSF and a new idea for the discovery and validation of pharmacodynamic substances in TCM.