Chebulagic Acid

The highly conserved N-terminal domain of polymerase acidic endonuclease (PA_N) makes it an attractive target for the development of anti-influenza virus drugs. This study developed an innovative multi-detector based at-line nanofractionation platform by coupling an HPLC system containing triple-detector (diode array detector (DAD), charged aerosol detector (CAD) and MS) with fluorescence resonance energy transfer (FRET)-based bioassays to systematically identify potential PA_N inhibitors from traditional Chinese medicine (TCM). A preliminary screening of 230 TCM extracts revealed an exceptional PA_N inhibition by Terminalia chebula Retz. (IC₅₀ = 0.88 μg/mL). A time-resolved FRET bioassay in 384 well-plates with parallelized MS/MS analysis enabled real-time acquisition of both biological activity profiles and chemical information from Terminalia chebula Retz. extracts. HPLC-DAD-CAD analysis further elucidated the correlation between the content and bioactivity of the identified potential PA_N-inhibitory compounds, addressing the limitations of traditional single UV-based detection. Remarkably, 31 bioactive compounds (including gallotannins, ellagitannins, and phenolic acids) were identified through this approach, with 75.9% exhibiting low abundance (CAD response < 1 pA·min), highlighting the unique capability of this platform in discovering low-abundant bioactive entities. Five verified compounds (ellagic acid, chebulinic acid, punicalagin, chebulagic acid, and 1,3,6-tri-O-galloyl-β-D-glucose) exhibited superior PA_N inhibition (IC₅₀ = 0.52-1.00 μM) in comparison to baloxavir marboxil (IC₅₀ = 10.83 ± 1.46 μM). A molecular docking study elucidated their crucial PA_N-binding interactions and fundamental mechanisms. Overall, this study not only identified several potential PA_N inhibitors from Terminalia chebula Retz., but also offered a scalable at-line nanofractionation strategy to reveal the content-activity correlation of complex TCM extracts.

Chebulae Fructus (CF) is commonly used to make juice, porridge, and also as dietary supplements owing to anti-inflammatory and antioxidant activities. However, astringency affects the palatability and compliance. During taste masking, it is essential to identify astringent mechanism to balance masking needs and efficacy maintenance. Pharmacophore was employed to screen potential astringent compositions, guiding the location of astringency. Qualitative and quantitative analyses were conducted using HPLC. Molecular docking was used to verify astringent components. Saliva proteins (SPs)-CF interaction was analyzed by SDS-PAGE, FT-IR, and fluorescence spectrum to explore astringent mechanism. Gallic acid, corilagin, 1,3,6-tri-O-galloylglucose, chebulagic acid, chebulinic acid, and ellagic acid were identified as key astringent substances. Hydrogen bonding and hydrophobic interaction facilitated the combination of CF with SPs. During interaction, SPs structures underwent folding, and partial α-helix and β-sheet structure transformed into β-turn. Overall, this study firstly elucidate the astringent mechanism of CF, establishing foundations for precise astringency masking.