An Anti-Inflammatory ent-Kaurane from the Stems of Annona squamosa that 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 16β,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 IC₅₀ values of 3.95 ± 0.68, 12.20 ± 2.16, and 12.52 ± 2.26 μM, 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.

Abbreviations

AC:adenylate cyclase

cAMP:cyclic adenosine 3′,5′-monophosphate

DPPH:1,1-diphenyl-2-picrylhydrazyl

FMLP:formyl-L-methionyl-L-leucyl-L-phenylalanine

O₂ ^·-:superoxide anion

PDE:phosphodiesterase

PKA:protein kinase A

LDH:lactate dehydrogenase

SOD:superoxide dismutase

WST-1:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt

Key words

Annona squamosa - Annonaceae - elastase - ent-kaurane - neutrophil - superoxide anion

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