An Anti-Inflammatoryent-Kaurane from the Stems ofAnnona squamosathat Inhibits Various Human Neutrophil Functions

Abstract

We have previously shown that 11 ent-kauranes isolated from the stems of Annona squamosa exhibited immunomodulating effects in leukocytes. In this study, a cellular model using isolated human neutrophils, which are important in the pathogenesis of rheumatoid arthritis, ischemia-reperfusion injury, chronic obstructive pulmonary disease, asthma and other inflammatory diseases, was established in order to elucidate the anti-inflammatory functions of 16beta,17-dihydroxy-ent-kauran-19-oic acid (1). Reactive oxygen species (ROS) and granule proteases produced by neutrophils contribute to the pathogenesis of inflammatory diseases. Compound 1 inhibited the generation of superoxide anion, the formation of ROS, and the release of elastase in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils in a concentration-dependent manner with IC (50) values of 3.95 +/- 0.68, 12.20 +/- 2.16, and 12.52 +/- 2.26 microM, respectively. The anti-inflammatory actions were not attributable to cytotoxicity because incubation of the neutrophils with 1 did not result in lactate dehydrogenase release. Compound 1 did not display antioxidant or superoxide anion-scavenging activity. Furthermore, neither subcellular NADPH oxidase activity nor cAMP-dependent pathways were altered by 1. Compound 1 significantly inhibited rapid calcium release from internal calcium stores induced by FMLP but not by thapsigargin. In summary, the presented results indicate that the inhibitory effects of 1 on respiratory burst and degranulation of human neutrophils are through the inhibition of cytosolic calcium mobilization, but not via the cAMP-dependent pathways.