Discrimination of the significance of astragalosides in Astragalus radix based on the in vivo exposure levels and pharmacokinetic characteristics of prototype saponins and their metabolites: A strategy for discovering and confirming quality markers

Astragali Radix (AR) is the dried root of Astragalus membranaceus var. mongholicus (Bge) Hsiao or A. membranaceus (Fisch.) Bge. AR has been consumed as a tonic in China for more than 2000 years because of its medicinal effects of invigorating the spleen and replenishing qi. Astragaloside Ⅰ (ASⅠ), astragaloside Ⅱ (ASⅡ), astragaloside Ⅲ (ASⅢ), astragaloside Ⅳ (ASⅣ), and cycloastragenol (CA) in AR exhibit diverse pharmacological activities. However, the current quality control methods mainly focus on ASⅣ while ignoring the contributions of other astragalosides. It cannot objectively and comprehensively reflect the quality of AR.

This study aimed to demonstrate the significance of the main astragalosides based on comprehensive in vivo exposure levels and pharmacokinetic characteristics of prototype saponins and their metabolites after oral administration of AR, providing a scientific rationale for establishing a multi-component quality control system using ASⅠ, ASⅡ, ASⅢ, and ASⅣ as quality markers (Q-markers) for AR.

An ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method was established for comprehensively evaluating the in vivo behaviors (pharmacokinetic parameters) of five principal ingredients of ASⅠ, ASⅡ, ASⅢ, ASⅣ and CA after oral administration of equimolar dose of alone component and water extract of AR (WEA) in rats. The importance of these saponin components as potential Q-markers was determined by the rate and extent of their conversion into CA in the body. Then, an ultra-high performance liquid chromatography tandem with evaporative light scattering detector (UHPLC-ELSD) method was established and applied to determine the content of ASⅠ, ASⅡ, ASⅢ, and ASⅣ in AR samples.

UHPLC-ESI-MS/MS method exhibits excellent specificity, accuracy, precision, and stability, with acceptable matrix effects and recovery rates. When rats were orally administered equimolar doses of ASⅠ, ASⅡ, ASⅢ, ASⅣ, CA and WEA, the most obvious characteristic was that they were mainly exposed as metabolite CA in blood. The characteristic pharmacokinetic parameters of CA converted from ASⅠ, ASⅡ, ASⅢ, ASⅣ, CA and WEA after oral administration equimolar dose of each were as: maximum peak concentration (C_max), 43.73, 20.58, 100.09, 44.20, 308.59 and 21.96 ng/mL, respectively; peak time (T_max), 11.33, 8.67, 6.67, 8.00, 1.00 and 9.33 h, respectively; and area under concentration-time curve (AUC_(0-t)), 600.08, 250.92, 1032.90, 464.18, 984.09 and 173.49 μg/L·h, respectively. The CA group demonstrated the most rapid systemic absorption and highest bioavailability of CA, ASⅢ showed optimal conversion kinetics and efficiency, followed closely by ASI which additionally exhibited prolonged pharmacological persistence. In contrast, ASⅣ and ASⅡ displayed inferior conversion efficiency with detectable prototype compounds in circulation. CA showed the highest plasma concentration after oral administration of WEA to rats, while little amounts of prototype ASⅡ and ASⅣ and no prototype ASⅠ or ASⅢ were observed, confirming that ASⅠ and ASⅢ undergo faster gastrointestinal conversion to CA compared to ASⅡ and ASⅣ. The abundance of ASⅠ, ASⅡ, ASⅢ, ASⅣ and CA in AR was determined by a validated UHPLC-ELSD method followed the order: ASⅠ > ASⅡ > ASⅣ ≥ ASⅢ.

This study systematically evaluated and compared the rates and efficiencies of ASⅠ, ASⅡ, ASⅢ, and ASⅣ converting to CA in vivo after oral administration equimolar dose of individual astragaloside in AR for the first time. ASⅠ and ASⅢ exhibited superior conversion kinetics and efficiency compared to ASⅣ and ASⅡ. Combining the transformation and exposure characteristics of astragalosides in vivo and their abundance in AR, ASⅠ, ASⅡ, ASⅢ, and ASⅣ can be used as potential multi-index markers to control the quality of astragalosides in AR. The in vivo ADME process characteristics of the main active components can be one of the effective strategies for screening potential objective Q-markers for Chinese medicinal materials or others.sss.