Quality and composition control of complex TCM preparations through a novel “Herbs-in vivo Compounds-Targets-Pathways” network methodology: The case of Lianhuaqingwen capsules

Lianhuaqingwen (LHQW) capsules, a Chinese patent medicine, consist of 13 herbal ingredients and are widely used for respiratory diseases. However, the complex composition of LHQW poses challenges in assessing its quality and consistency. In this study, a comprehensive network of LHQW was constructed by integrating Digital RNA with pertUrbation of Genes (DRUG)-seq, RNA sequencing, and pharmacodynamic data. This enables rapid and systematic screening of compounds in LHQW that exhibit high-exposure in vivo and significant activity potential, serving as potential quality control markers. In details, DRUG-seq was employed to evaluate gene expression alterations in peripheral blood mononuclear cells derived from healthy volunteers. Ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS) was used to analyze the components of LHQW in rats, identifying a total of 505 compounds. Additionally, absorption, distribution, metabolism, and excretion (ADME) profiles were plotted for 27 primary components of LHQW. And an HPLC-MS/MS method quantified 46 compounds from LHQW, with 15 of them identified as potential quality markers with high exposure levels. These markers exhibited significant inhibitory effects on lipopolysaccharide (LPS)-induced pneumonia in mice, with mechanisms predicted by RNA-seq and verified by RT-qPCR. In summary, this study successfully constructed an "Herbs- in vivo Compounds-targets-pathways" network, offering novel insights into the mechanisms of LHQW and establishing a foundation for enhancing quality control measures. • Based on in vivo ADME, combined with DRUG-seq, RNA-seq technology, in vivo and in vitro drug efficacy verification, a "Herbs- in vivo Compounds-targets-pathways" network diagram of LHQW was constructed. • From the holistic view of traditional Chinese medicine, compounds with high-exposure in vivo and activity potential in LHQW were systematically screened as quality control markers, based on the in vivo perspective, providing reference for improving the quality control standards for LHQW. • A comprehensive ADME profile analysis of 27 LHQW compounds in rats was completed, and HPLC-MS/MS quantitative analysis method for 46 compounds was established to clarify the pharmacokinetic characteristics of the main exposed components of LHQW in vivo .