Acteoside (ACT) is also known as verbascoside and widely exists in various traditional Chinese medicine of the Verbenaceae, Labiatae, Xanthaceae and Miscanthaceae families, especially in the leaves of Rehmannia glutinosa (Gaetn.) Libosch. ex Fisch. & C.A. Mey. The leave of Rehmannia glutinosa (Dihuangye) belongs to heart, liver and kidney meridians, according to its first record in the Dietetic Materia Medica. Li Shi-zhen had said “the essence was in the leaf, not all in the root” in the Compendium of Materia Medica. Dihuangye has the function of clearing heat, replenishing qi and activating blood, nourishing yin and tonifying kidney. In the Important Prescriptions Worth a Thousand Gold for Emergency, Dihuangye was used to treat sore carbuncle. Base on the principle of comprehensive utilization, we had fully developed Dihuangye. Dihuangye total glycosides were extracted from Dihuangye and marketed in China in capsule form as a new Class II Chinese medicine by our team. Clinical trials have confirmed the safety and efficacy in reducing proteinuria (Qiu et al., 2014). ACT is the main ingredient of Dihuangye total glycosides capsule, accounting for about 35%. Therefore, it is important to study the metabolism and mechanism of ACT in treating chronic glomerulonephritis (CGN). ACT is a naturally occurring glycoside with β-glucose as the parent nucleus, and it contains ester acyl and oxyglycoside bonds and a glycosidic linkage between caffeic acid (CA) and 3,4-dihydroxyphenylethanol (DOPE). ACT has been shown to have various biological activities in vitro and in vivo, such as anti-inflammatory (Chen et al., 2009; Speranza et al., 2010), antitumor (Song and Sim, 2009), antihepatotoxic (Xiong et al., 1998), uric acid reducing (Huang et al., 2008) and neuroprotective activities (Burgos et al., 2020; Sheng et al., 2002). It has been chosen as a marker for the quality control of herbal medicines (Qi et al., 2012). Furthermore, the degradation products of ACT, especially the CA and DOPE groups, possessed evident bioactivities such as antioxidant (Gordon et al., 2001) and neuroprotective effects (Koo et al., 2006; Schaffer et al., 2007).

The identification of metabolites of ACT in vivo is indispensable for illustrating its metabolic patterns and proposed mechanisms. In previous studies, ACT was usually considered a prodrug metabolized in the gastrointestinal tract and liver after administration to produce active metabolites or intermediates for pharmacological action (Wu et al., 2009). The metabolic behaviors of ACT in vivo have been reported, and the metabolites are concentrated on less polar compounds (Su et al., 2016). However, its metabolic behavior has not been reported in model animals, which might present different results relative to normal animals. An in-depth study of the metabolic profile of ACT by the oral route is essential for both physiological and pathological conditions. Data obtained from pathological conditions might be more meaningful than those obtained from normal conditions for clinical applications.

A previous study focused on the renoprotective effect and possible mechanisms of ACT in passive Heymann nephritis (PHN) rats. The effects on adhesion molecule and inflammatory cytokine production, vasoactive substance balance, platelet aggregation, transforming growth factors and matrix proteins were observed, which provided a research basis for the development of ACT as a drug for the treatment of chronic nephritis.

The metabolism of ACT was systematically investigated by ultra high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF MS). Q/TOF MS has recently become a powerful and reliable analytical technique for metabolite identification (Wang et al., 2018, 2021). In the present study, comprehensive metabolites of ACT in rat plasma, feces and urine after oral administration were studied. Furthermore, metabolites in urine samples were validated after ACT was administered to pathological models, and the results would provide theoretical support for the clinical treatment of ACT in CGN. According to the metabolic analysis, ACT and its two metabolites, DOPE and 3,4-dihydroxyphenylacetic acid (DOPAC), were selected to intervene in glomerular podocytes induced by high glucose (HG) to investigate the cell morphology and the expression of nephrin. Based on the analysis of ingredients in vivo, the bioactive constituents and potential therapeutic targets in the treatment of CGN were investigated by using network pharmacological analysis and molecular docking.