Anti-inflammatory effects of Glehnia littoralis ethyl acetate extract and its active compound phellopterin via inhibition of N-acylethanolamine acid amidase

Ethnopharmacological relevance: In East Asia, the root of Glehnia littoralis (J.G.Cooper) F.Schmidt ex Miq. is traditionally used to relieve "yin-deficiency lung dryness," nourishing yin, moistening the lungs, and promoting the generation of body fluids. It is commonly incorporated into decoctions to relieve chronic inflammatory symptoms. However, the primary active constituents and the molecular mechanisms responsible responsible for its anti-inflammatory effects remain insufficiently defined.

Aim of the study: The present study was designed to evaluate the anti-inflammatory potential of the ethyl acetate fraction of G. littoralis (EA) and to identify phellopterin and cnidilin as key constituents responsible for inhibition N-acylethanolamine acid amidase (NAAA).

Materials and methods: Five crude fractions were obtained from G. littoralis through systematic extraction and bioactivity-guided fractionation. EA was identified as the most active fraction by parallel screening using an NAAA inhibition assay and an LPS-induced RAW264.7 cells inflammation model. After confirmation its efficacy in cellular and animal models, the chemical constituents of EA were profiled by UPLC-Q-TOF MS, and two major compounds were selected for further evaluation. These candidates were screened using the same NAAA and cellular assays to identify the most potent anti-inflammatory compound, whose activity was subsequently validated in both in vitro and in vivo models.

Results: In LPS-stimulated RAW264.7 cells, EA (25-100 μg/mL) inhibited NAAA activity (IC₅₀ = 27.98 μg/mL), reduced the production of NO, IL-6 and TNF-α, and increased endogenous levels of PEA and OEA.In vivo, EA (30-100 μg) topical administration significantly attenuated carrageenan-induced paw edema and croton oil-induced ear edema, accompanied by decreased tissue levels of IL-6 and TNF-α. UPLC-Q-TOF MS analysis identified phellopterin as a principal constituent of EA. Subsequent in vitro and in vivo evaluations demonstrated that phellopterin exhibits pronounced NAAA inhibitory and anti-inflammatory activities. Specifically, phellopterin (10-30 μg) ameliorated carrageenan-induced paw edema and croton oil-induced ear edema, and reduced tissue IL-6 and TNF-α levels, thereby confirming its role as a pivotal anti-inflammatory constituent of the EA fraction.

Conclusions: This study demonstrates that the ethyl acetate extract of G. littoralis and its active compound, phellopterin, exert anti-inflammatory effects by inhibiting NAAA, elevating the anti-inflammatory N-acylethanolamines OEA and PEA, and suppressing pro-inflammatory cytokines in both cellular and murine models. These findings provide pharmacological support for the traditional use of G. littoralis and suggest that phellopterin represents a promising lead compound for the development of NAAA-targeted anti-inflammatory agents.

Ear edema; Endocannabinoid system; Glehnia littoralis; NAAA; OEA; PEA; Phellopterin.