Screening and Determination of Biomarkers and Their Associated Active Components for Enhanced Therapeutic Effect of Exocarpium Citri Grandis–Folium Ginkgo Couplet on Liver Injury Caused by Hyperlipidemia via UPLC-TOF-MS and UPLC-MS

Caihui Liu,1,2,* Guangying Wu,1,2,* Xingqin Wei,1,2 Guanlin Xiao,1,2 Zhao Chen1,21The Fifth College of Clinic Medicine, Guangzhou University of Chinese Medicine, Guangzhou, 510095, People’s Republic of China; 2Guangdong Provincial Key Laboratory of Research and Development in Traditional Chinese Medicine, Guangzhou, 510095, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhao Chen, Email cz547567@163.comIntroduction: The combination of Exocarpium Citri Grandis–Folium Ginkgo (E–GC) demonstrates superior therapeutic effects on liver injury caused by hyperlipidemia compared with individual use; however, accurate and quantifiable indicators for evaluating the efficacy and quality of the couplet remain lacking. Reliance solely on changes in certain differential metabolites or the bioavailability of chemical components is insufficient to confirm the superior efficacy of the couplet.Methods: In this study, a rat model of liver injury caused by hyperlipidemia was used to verify the enhanced efficacy of E-GC. Subsequently, network pharmacology combined with molecular biology validation was applied to identify the primary regulatory targets. The results were integrated with UPLC-TOF-MS, UPLC-MS and database analyses to identify biomarkers indicating the enhanced efficacy of E-GC, as well as serum concentrations of active components in E-GC to quantitatively evaluate the effect of the couplet on their bioavailability. Finally, correlation statistical analyses were conducted on the above-mentioned data to confirm that the identified biomarkers and significantly altered active components can characterize the enhanced efficacy of the couplet.Results: Metabolomics analysis identified 59 related metabolites, among which arachidonic acid, choline, deoxycholic acid, N-acetylserotonin, and cholic acid significantly increased in the liver following E-GC treatment, with similar changes observed in serum levels (p< 0.05). A total of 36 components were identified, apigenin, naringin, and ginkgolide A/B/C exhibited significantly increased bioavailability after combination (p< 0.05). These changes were closely associated with the improved regulation of PXR-FXR expression by E-GC compared with individual use, and E-GC also exerted strong regulatory effects on downstream targets, such as LXR, FGF1, ABCB1b, and ERK.Conclusion: Through the above analyses, in order to solve the problem of lacking accurate and quantifiable indexes to evaluate the effect of TCM couplet, a method was successfully established for evaluating the enhanced efficacy of E-GC in treating liver injury caused by hyperlipidemia, and the principal biomarkers and quality control indicators based on active components were identified.Keywords: exocarpium citri grandis–folium ginkgo couplet, biomarkers for enhancing drug efficacy, associated active ingredients, UPLC-TOF-MS, UPLC-MS-MS