Anti-angiogenic effects and mechanisms of zerumin A from Alpinia caerulea

Alpinia caerulea (R.Br.) Bentham, a perennial herb growing in tropical and subtropical Australia, is used as a flavouring spice and a ginger substitute. Its fruit has been used as indigenous food among the aboriginal Australians; 95% ethanol extracts of the dried fruits, leaves, rhizomes and roots of this plant were investigated in a zebrafish model by quantitative endogenous alkaline phosphatase assay. Only the fruit extract showed potential anti-angiogenic effect, inhibiting vessel formation by 25% at 20 μg/ml. Two diterpenoids were isolated and identified as zerumin A and (E)-8(17),12-labdadiene-15,16-dial. Zerumin A, which had mainly accumulated in the fruits and bearing a carboxylic group, could dose-dependently inhibit vessel formation, in both wild-type and Tg(fli1a:EGFP)y1 zebrafish embryos. The semi-quantitative reverse transcription polymerase chain reaction assay on wild type zebrafish embryos suggested that zerumin A affected multiple molecular targets related to angiogenesis. Further investigation, by human umbilical vein endothelial cell assays, revealed that zerumin A specifically inhibited the proliferation and migration steps, to prevent angiogenesis progress.