Two triterpenoids, cucurbitacins B and D, have been isolated from seeds of Iberis umbellata (Cruciferae) and shown to be responsible for the antagonistic activity of a methanolic extract of this species in preventing the 20-hydroxyecdysone (20E)-induced morphological changes in the Drosophila melanogaster BII permanent cell line. With a 20E concentration of 50 nM, cucurbitacins B and D give 50% responses at 1.5 and 10 microM respectively. Both cucurbitacins are able to displace specifically bound radiolabelled 25-deoxy-20-hydroxyecdysone (ponasterone A) from a cell-free preparation of the BII cells containing ecdysteroid receptors. The Kd values for cucurbitacins B and D (5 and 50 microM respectively) are similar to the concentrations required to antagonize 20E activity with whole cells. Cucurbitacin B (cucB) prevents stimulation by 20E of an ecdysteroid-responsive reporter gene in a transfection assay. CucB also prevents the formation of the Drosophila ecdysteroid receptor/Ultraspiracle/20E complex with the hsp27 ecdysteroid response element as demonstrated by gel-shift assay. This is therefore the first definitive evidence for the existence of antagonists acting at the ecdysteroid receptor. Preliminary structure/activity studies indicate the importance of the Delta23-22-oxo functional grouping in the side chain for antagonistic activity. Hexanorcucurbitacin D, which lacks carbon atoms C-22 to C-27, is found to be a weak agonist rather than an antagonist. Moreover, the side chain analogue 5-methylhex-3-en-2-one possesses weak antagonistic activity.

The occurrence and levels of phytoecdysteroids in the seeds and other parts of plants grown from the seeds of 180 randomly selected plant species were assessed and compared. Ecdysteroids are frequently detectable in leaves and flowers, but less so in stems, roots and seeds. The seeds of 290 species were assessed for the presence of hydrolysable ecdysteroid conjugates. Low levels of conjugates were detected in a significant number of species, large amounts being present only when levels of free ecdysteroids were high. Individual plants of Arabidopsis thaliana were assessed for the presence of phytoecdysteroids. While plants of this species are generally ecdysteroid negative, individual plants in the population accumulate low levels of ecdysteroids. Extracts of seeds of 50 "ecdysteroid-negative" species were concentrated and partially purified to determine if they possess ecdysteroids at levels below the usual detection levels. Ecdysteroids were detectable by radioimmunoassay in almost all of these concentrated samples. Thus, all lines of evidence point to the conclusion that all species of plants have the capacity to produce at least low levels of phytoecdysteroids. This has important implications for the protection of crop species through enhancing ecdysteroid levels by breeding/genetic modification strategies.

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