Four new piperidine amide alkaloids from Piper longum fruits and their anti-inflammatory activities.

Piper longum L., a widely recognized Chinese traditional spice, is extensively used as dietary flavoring agent in Southeast Asia due to its unique taste. As a dual-purpose resource for both food and medicine, the fruits of P. longum have garnered significant attention for their diverse chemical constituents and notable physiological activities. In this study, the phytochemical investigation of the fruits of P. longum resulted in the isolation and identification of 20 structurally diverse piperidine amide alkaloids, including 4 new (1a/1b-4a/b) and 16 known analogues (5-20). The enantiomeric mixture of 1a/1b was successfully separated using a chiral column with acetonitrile-water as eluents, while chiral HPLC analysis confirmed that 4 existed as an enantiomeric mixture (4a/4b) without achieving separation. Their structures were elucidated through comprehensive spectroscopic data analysis (NMR, HRESIMS, IR, UV), electronic circular dichroism (ECD) and by comparison with literature data. Biological evaluations revealed that compounds 2, 3, 16, and 17 exhibited significant inhibitory effects on NO secretion in LPS-stimulated RAW264.7 cells, with IC50 values ranging from 9.9 to 15.76 μM, which were better than hydrocortisone (IC50 = 34.26 ± 2.20 μM). Furthermore, Western blot and real-time PCR assays demonstrated that 2 and 17 markedly downregulated the mRNA levels and the protein expression of iNOS. Notably, 17 partially inhibited the phosphorylation of p65 and reduced the protein expression of TLR4. Thus, the discovery of structurally diverse anti-inflammatory piperidine amide alkaloids from the fruits of P. longum in this study could benefit the further development and utilization of this plant.