 PERGAMON Phytochemistry 49 "0888# 0012Ð0017 9920!8311:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[ PII]S9920!8311"87#99542!9 Ecdysteroid antagonists "cucurbitacins# from Physocarpus opulifolius "rosaceae# Satyajit D[ Sarker a \ Pensri Whiting a \ Vladimir S ³ ik b \ Laurence Dinan a\  a Hatherly Laboratories\ Department of Biological Sciences\ University of Exeter\ Prince of Wales Road\ Exeter\ Devon EX3 3PS\ UK b Department of Chemistry\ University of Exeter\ Stocker Road\ Exeter\ Devon EX3 3QD\ UK Received in revised form 03 September 0887 Abstract Methanolic extracts of seeds of 3 species in the genus Physocarpus antagonise the action of the insect steroid hormone 19! hydroxyecdysone on a Drosophila melanogaster permanent cell line[ The active components in the extract of P[ opulifolius "ninebark# have been identi_ed as cucurbitacin D\ cucurbitacin F and 2!epi!isocucurbitacin D[ The potencies of the individual cucurbitacins have been determined as 4×09 −6 \ 7×09 −6 and 6×09 −5 M\ respectively "versus 4×09 −7 M 19!hydroxyecdysone#[ The distribution of antagonistic activity in plants of P[ opulifolius has been assessed and HPLC:bioassay has been used to determine the chromato! graphic pro_les of antagonist activity in parts of the growing plant[ Þ 0888 Elsevier Science Ltd[ All rights reserved[ Keywords] Physocarpus opulifolius^ Rosaceae^ Ninebark^ Cucurbitacin D^ 2!epi!Isocucurbitacin D cucurbitacin F^ Ecdysteroid antagonist^ Chem! otaxonomy 0[ Introduction Many plant secondary compounds have been shown to a}ect insect growth\ development and:or fecundity and are consequently believed to play a role in the deter! rence of insect predators[ However\ the molecular modes of action of very few of these compounds have been elucidated[ One class of secondary compounds which have been postulated to contribute to the deterrence of non!adapted phytophagous insects are the phyto! ecdysteroids\ which are analogues of insect steroid hor! mones[ Over 049 structurally!related phytoecdysteroids have been isolated from a wide variety of plant species "Lafont + Wilson\ 0885#[ All phytoecdysteroids act as hormone agonists of the insect ecdysteroid receptor "Dinan\ Hormann\ + Fujimoto\ 0887#[ There is growing interest in the receptor as a target for the development of invertebrate!speci_c pest control agents[ To exploit this target it is important to characterise the ligand!binding site and to identify novel agents interacting speci_cally with the receptor\ either as agonists "such as the phyto! ecdysteroids# or as antagonists[ We have screened 0 Present address] Institute of Grassland and Environmental Research\ Xenova Discovery at IGER\ Plas Gogerddan\ Aberystwyth\ Ceredigion\ SY12 2EB\ UK[  Corresponding author[ Tel[] ¦33!0281!153!594^ fax] ¦33!0281! 153!557^ e!mail] l[n[dinanÝexeter[ac[uk[ extracts of ×3999 species of plants for the presence of ecdysteroid receptor agonists and antagonists using a simple and e}ective bioassay "Cle ment\ Bradbrook\ Lafont\ + Dinan\ 0882^ Dinan\ 0884#[ One of the genera possessing distinct antagonistic activity was Physocarpus[ In this paper\ we report the isolation of the active con! stituents in P[ opulifolius "L[# Maxim[\ commonly known as ninebark[ Using bioassay!guided HPLC puri_cation\ we isolated the active compounds and identi_ed them as three cucurbitacins] cucurbitacin D "0#\ cucurbitacin F "1# and 2!epi!isocucurbitacin D "2#[ Cucurbitacins are reported for the _rst time from P[ opulifolius and have only occasionally been isolated from members of the Rosaceae previously[ 1[ Results and discussion Although the presence of ecdysteroid agonists "phyto! ecdysteroids# in plants was _rst demonstrated over 29 years ago\ no de_nitive ecdysteroid antagonists had been identi_ed until recently\ when it was demonstrated that cucurbitacins from Iberis umbellata "Cruciferae "Dinan et al[\ 0886## and Cercidiphyllum japonicum "Cercidi! phyllaceae "Sarker\ Whiting\ Sik\ + Dinan\ 0886## and certain withanolides from Iochroma gesnerioides "Sol! anaceae "Dinan\ Whiting\ Alfonso\ + Kapetanidis\ 0885## were active[ Further species showing signi_cant