Characterization Of Chemical Constituents And Study On Activities Of Gynura Divaricata(L.)DC.

Objective:To systematically characterize the chemical constituents of Gynura divaricata（L.）DC.and study their biological activities and mechanism of action.Methods:（1）An ultra-high performance liquid chromatography tandem high-resolution mass spectrum combined with molecular networking were used to characterize chemical constituents of Gynura divaricata（L.）DC.（2）Enzyme inhibitory activity tests and in vitro cell experiments were utilized to evaluate the biological activities of Gynura divaricata（L.）DC.（3）Molecular docking,network pharmacology and transcriptomics were employed to explore the mechanism.Results:（1）Diprotin A（a tripeptide DPP-IV inhibitor）and tripeptide analogs were first reported.Twenty-five diprotin A analogs were characterized.（2）A mixture of twenty-five diprotin A analogs exhibited an IC₅₀ of 0.40mg/m L for DPP-IV.Molecular docking results also confirmed the interactions between the tripeptide analogs and DPP-IV mainly via H-bonds and hydrophobic interactions.（3）A total of 75 sesquiterpenoids（rupestonic acid analogs）were tentatively characterized in non-alkaline ingredients（GD-NAIs）,in which 61sesquiterpenoids were reported for the first time.（4）GD-NAIs significantly inhibited proliferation and promoted apoptosis of CML cells.The anti-CML activity of GD-NAIs was stronger than GD-E.GD-NAIs had low toxicity on normal liver cells.（5）Combining the network pharmacology and RNA sequencing,we demonstrated that GD-NAIs exhibited anti-CML activity by inhibiting PD-L1expression and the PD-1 checkpoint pathway in cancer,PI3K/AKT,JAK/STAT,TGF-β,estrogen,Notch and Wnt signaling pathways.Conclusions:（1）There are tripeptide analogs and sesquiterpenoids in Gynura divaricata（L.）DC.（2）Tripeptide analogs isolated from Gynura divaricata（L.）DC.were characterized as DPP-IV inhibitors,which exhibited an IC₅₀ of 0.40 mg/m L for DPP-IV.This finding demonstrates that Gynura divaricata（L.）DC.would be developed as hypoglycemic natural products in the future.（3）GD-NAIs exhibited in vitro anti-CML activity.