Investigation of constituents from Cinnamomum camphora (L.) J. Presl and evaluation of their anti-inflammatory properties in lipopolysaccharide-stimulated RAW 264.7 macrophages

Abstract

Ethnopharmacology relevanceCinnamomum camphora (L.) J. Presl has been used for the traditional medicine as a therapeutic agent of inflammation-related diseases, including sprains, rheumatic arthritis, abdominal pain, cough and bronchitis, for a long history. The aim of the present study was to illustrate anti-inflammatory substances of C. camphora and their mechanism of action, and to establish the correlations between chemical constituents and traditional uses of this plant. Materials and methodsChemical constituents were purified by chromatographic methods, and their structures were established based on spectroscopic analysis. Lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages was adopted for evaluating the anti-inflammatory activity in vitro. The nitric oxide (NO) production assay and nuclear factor kappa B (NF-κB) dual luciferase reporter assay were used to screen anti-inflammatory constituents. The mRNA and protein levels of inflammation-related cytokines and enzymes were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR), immunoblot analysis, and enzyme linked immunosorbent assay (ELISA), respectively. ResultsTwenty-five constituents were isolated from the EtOH extract of C. camphora. Eight constituents, covering phenylpropanoid (7), lignans (10 and 22), flavonoids (16–18), coumarin (21), and terpenoid (24) significantly inhibited LPS-stimulated NO production with maximum inhibition rates (MIRs) of ≥ 80%, and thus were verified to be the anti-inflammatory substances of this ethnomedical plant. (+)-Episesaminone (SMO, 22) and 3S-(+)−9-oxonerolidol (NLD, 24) blocked NF-κB activation via inducing IκBα expression. Moreover, SMO and NLD inhibited productions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2), and alleviated increased mRNA and protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and matrix metallopeptidase-9 (MMP-9) in LPS-stimulated RAW 264.7 macrophages. ConclusionsThe ethnomedical use of C. camphora for the treatment of inflammation-related diseases was attributed to the combined in vitro anti-inflammatory activities of phenylpropanoid, lignan, flavonoid, coumarin, and terpenoid. SMO and NLD were found to be new molecules with in vitro anti-inflammatory activities, which are achieved by inhibiting NF-κB regulated inflammatory response.