Isolation and characterization of sesquiterpenes from Arecophila saccharicola YMJ96022401 with NO production inhibitory activity

Sesquiterpenes, arecoic acids A-F and arecolactone, were isolated from the ethyl acetate extracts of the fermented broth of Arecophila saccharicola YMJ96022401 along with two known analogues 1,7α,10α- trihydroxyeremophil-11(13)-en-12,8-olide and 1,10α,13-trihydroxyeremophil- 7(11)-en-12,8-olide. Their structures were elucidated on the basis of spectroscopic data analyses. The inhibitory effects of all of these compounds on nitric oxide (NO) production in lipopolysaccharide (LPS, 200 μg/mL)-activated murine macrophage RAW264.7 cells were also evaluated. Among these compounds, 1,7α,10α-trihydroxyeremophil-11(13)-en-12,8-olide significantly inhibited NO production without any cytotoxicity, and its average maximum inhibition (E_max) at 100 μM and median inhibitory concentration (IC₅₀) were 85.7% ± 0.8% and 16.5 ± 1.0 μM, respectively. Arecolactone was the most potent, with the E_max at 12.5 μM and IC₅₀ being 94.7% ± 0.8% and 1.32 ± 0.1 μM, respectively, but displayed cytotoxicity at considerable higher concentrations than 25 μM. Analyses of Western blotting indicated that arecolactone (0.8-12.5 μM) inhibited induction of inducible NO synthase (iNOS) by LPS, which involved suppression of NF-κB activation and the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) in activated RAW 264.7 cells. In addition, arecolactone concentration-dependently prevented the vascular hyporeactivity to phenylephrine induced by LPS (300 ng/mL) through iNOS pathway in isolated rat thoracic aortic rings. These results indicated that both of these naturally occurring iNOS inhibitors may provide a rationale for the potential anti-inflammatory effect of A. saccharicola YMJ96022401.