Ramulus Cinnamomi essential oil exerts an anti-inflammatory effect on RAW264.7 cells through N-acylethanolamine acid amidase inhibition

Ethnopharmacological relevance: Ramulus Cinnamomi, the dried twig of Cinnamomum cassia (L.) J.Presl., is a traditional Chinese medicine (TCM) with anti-inflammatory effects. The medicinal functions of Ramulus Cinnamomi essential oil (RCEO) have been confirmed, although the potential mechanisms by which RCEO exerts its anti-inflammatory effects have not been fully elucidated.

Aim of the study: To investigate whether N-acylethanolamine acid amidase (NAAA) mediates the anti-inflammatory effects of RCEO.

Materials and methods: RCEO was extracted by steam distillation of Ramulus Cinnamomi, and NAAA activity was detected using HEK293 cells overexpressing NAAA. N-Palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), both of which are NAAA endogenous substrates, were detected by liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The anti-inflammatory effects of RCEO were analyzed in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and the cell viability was measured with a Cell Counting Kit-8 (CCK-8) kit. The nitric oxide (NO) in the cell supernatant was measured using the Griess method. The level of tumor necrosis factor-α (TNF-α) in the RAW264.7 cell supernatant was determined using an enzyme-linked immunosorbent assay (ELISA) kit. The chemical composition of RCEO was assessed by gas chromatography-mass spectroscopy (GC-MS). The molecular docking study for (E)-cinnamaldehyde and NAAA was performed by using Discovery Studio 2019 software (DS2019).

Results: We established a cell model for evaluating NAAA activity, and we found that RCEO inhibited the NAAA activity with an IC₅₀ of 5.64 ± 0.62 μg/mL. RCEO significantly elevated PEA and OEA levels in NAAA-overexpressing HEK293 cells, suggesting that RCEO might prevent the degradation of cellular PEA and OEA by inhibiting the NAAA activity in NAAA-overexpressing HEK293 cells. In addition, RCEO also decreased NO and TNF-α cytokines in lipopolysaccharide (LPS)-stimulated macrophages. Interestingly, the GC-MS assay revealed that more than 93 components were identified in RCEO, of which (E)-cinnamaldehyde accounted for 64.88%. Further experiments showed that (E)-cinnamaldehyde and O-methoxycinnamaldehyde inhibited NAAA activity with an IC₅₀ of 3.21 ± 0.03 and 9.62 ± 0.30 μg/mL, respectively, which may represent key components of RCEO that inhibit NAAA activity. Meanwhile, docking assays revealed that (E)-cinnamaldehyde occupies the catalytic cavity of NAAA and engages in a hydrogen bond interaction with the TRP181 and hydrophobic-related interactions with LEU152 of human NAAA.

Conclusions: RCEO showed anti-inflammatory effects by inhibiting NAAA activity and elevating cellular PEA and OEA levels in NAAA-overexpressing HEK293 cells. (E)-cinnamaldehyde and O-methoxycinnamaldehyde, two components in RCEO, were identified as the main contributors of the anti-inflammatory effects of RCEO by modulating cellular PEA levels through NAAA inhibition.

(E)-cinnamaldehyde (PubChem CID: 637511); (E)-cinnamic alcohol (PubChem CID: 308); (Z)-3-Phenylacrylaldehyde (PubChem CID: 6428995); 3-Phenyl-1-propanol (PubChem CID: 31234); Acetophenone (PubChem CID: 7410); Anti-inflammation; Benzaldehyde (PubChem CID: 240); Benzenepropanal (PubChem CID: 7707); Bisabolol (PubChem CID: 1549992); Butylated Hydroxytoluene (PubChem CID: 31404); Coumarin (PubChem CID: 323); Epi-beta-acorenol (PubChem CID: 91747406); HEK293T-hNAAA cell; Hexanedioic acid, bis (2-ethylhexyl) ester (PubChem CID: 7641); NAAA inhibitors; O-Menth-2-ene (PubChem CID: 521885); O-methoxycinnamaldehyde (PubChem CID: 641298); PEA; PPAR-α; Phenylethyl alcohol (PubChem CID: 6054); Phthalic acid, hept-3-yl isobutyl ester (PubChem CID: 91719723); Ramulus Cinnamomi essential oil; Spathulenol (PubChem CID: 92231); T-muurolol (PubChem CID: 3084331); β-Guaiene (PubChem CID: 15560252); γ-Himachalene (PubChem CID: 577062).