Integrating in vivo metabolism, pharmacokinetics, and dual-indicator scoring for PK-marker selection of Celosiae Semen saponins.
Abstract
The total saponins from Celosiae Semen (TSCS) show hepatoprotective and lipid-lowering effects against metabolic dysfunction-associated steatotic liver disease (MASLD). However, their in vivo metabolic fate and holistic pharmacokinetic (PK) features remain largely uncharacterized. In this study, an integrated PK-marker screening strategy was established by combining in vivo metabolic profiling, area under the curve (AUC)-weighted composite PK analysis, and a dual-indicator scoring model that weights both systemic exposure and in vitro lipid-lowering activity. A total of 25 saponins were characterized in TSCS by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS), and 107 TSCS-related substances (8 prototypes and 99 metabolites) were tentatively characterized in rats, with sequential deglucosylation tentatively characterized as the predominant metabolic pathway. Among the characterized saponins, five representative saponins (celosin J, H, I, K, and L) were selected for bioactivity evaluation and found to concentration-dependently reduced lipid accumulation in palmitic acid (PA)-induced HepG2 cells. Subsequently, a validated liquid chromatography coupled with triple quadrupole tandem mass spectrometry (LC-QQQ-MS/MS) method was developed for simultaneous plasma quantification of these five bioactive saponins, revealing marked exposure differences among them (AUC₀-∞: 40.95-359.05 μg·min/mL). An AUC‑weighted integration model was established to derive composite PK indices (T₁/₂ 428 min, Cₘₐₓ 1.24 μg/mL, AUC₀-∞ 215.45 μg·min/mL) that adequately reflect the holistic systemic behavior of TSCS. On the basis of systemic exposure and in vitro activity, the dual-indicator scoring model identified celosin I as the optimal PK-marker (score 0.910) and celosin H as a complementary marker (score 0.616). In a fructose-induced zebrafish MASLD model, both compounds dose-dependently reduced body length, body weight, hepatic lipid deposition, and hepatic total cholesterol (TC), triglyceride (TG), and free cholesterol (FC) levels and upregulated the expression of peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1α (CPT-1α), and acyl-CoA oxidase 1 (ACOX1), preliminarily supporting the in vivo relevance of the selected markers. This integrated strategy mitigates the limitations of single-indicator screening approaches and provides a scientific basis for the quality control and standardization of TSCS.