Phytochemistry\ Vol[ 38\ No[ 1\ pp[ 312Ð318\ 0887 Þ 0887 Elsevier Science Ltd[ All rights reserved Pergamon Printed in Great Britain \ 9920Ð8311:87:,Ðsee front matter PII] S9920Ð8311"87#99078Ð6

VOLATILE MONOTERPENES IN AND P[ RUDERALE "#] IDENTIFICATION\ LOCALIZATION AND INSECTICIDAL SYNERGISM WITH a!TERTHIENYL

IN HONOUR OF PROFESSOR G[ H[ NEIL TOWERS 64TH BIRTHDAY

GABRIEL GUILLET\$ ANDREł BEłLANGER% and JOHN T[ ARNASON$

$Department of Biology\ University of Ottawa\ 29 Marie Curie\ Ottawa\ Ontario\ K0N 5N4\ Canada and %Agriculture Canada\ St!Jean sur Richelieu\ Quebec\ J2B 2E5\ Canada[

"Received in revised form 1 October 0886#

Key Word Index*Porophyllum `racile^ ^ asteraceae^ monoterpene^ a!terthienyl^ synergism[

Abstract*Volatiles occurring in Porophyllum `racile and P[ ruderale "Asteraceae# were identi_ed in order to assess their role in integrated chemical defences against insects[ Headspace and steam distillation techniques yielded a total of 01 mono! and sesquiterpenes and fatty acid derivatives[ The glandular secretory cavities present on leaves of P[ ruderale were large enough to permit direct sampling and were found to be particularly rich in volatile monoterpenes "62[5) of the integrated FID trace#[ Using reduction of relative growth rate of third instar Ostrinia nubilalis "Lepidoptera] Pyralidae# larvae as an index of insecticidal activity\ the volatiles released from the secretory cavities located on the leaves of P[ ruderale had no signi_cant e}ect alone\ but they synergized the e}ects of a!terthienyl\ a toxic light!activated secondary compound also present in P[ `racile and P[ ruderale[ This synergistic interaction was shown to be related to an enhanced accumulation of the a! terthienyl in O[ nubilalis larvae when they were exposed to the volatiles emitted from the foliar secretory cavities of P[ ruderale[ Þ 0887 Elsevier Science Ltd[ All rights reserved

INTRODUCTION also con_rmed under laboratory conditions that the volatile compounds emitted from the foliar GSCs of The genus Porophyllum "Asteraceae#\ which occurs in both P[ ruderale and P[ `racile exert a repellent activity an area extending from the southwest of the United against adults of the red!legged grasshopper\ Melano! States to ð0\ 1Ł\ is characterized by the plus femurrubrum femurrubrum "Orthoptera] Acri! presence of translucent glandular secretory cavities didae#[ "GSCs# that are located along the leaf margin and\ to Di}erent monoterpenes\ sesquiterpenes and photo! a less extent\ scattered throughout the lamina ð2Ł[ The toxic thiophenes\ have been identi_ed in the genus lumen of these GSCs has a lysigenous origin and is Porophyllum ð5Ð01Ł[ It is\ however\ not known which delimited by a multilayered epithelium ð3Ł[ In a pre! insecticidal compounds typically occur in the foliar vious study we reported that the GSCs were also an GSCs of Porophyllum spp[ since the chemicals important feature that confers resistance against reported in the above studies were in most cases insect herbivory in Porophyllum `racile "Jacq[# Cass[ Var[ macrocephalum "DC[# and Porophyllum ruderale extracted from tissues of either entire aerial parts\ Benth[ "Asteraceae# ð4Ł[ Under _eld conditions\ indi! roots or whole [ viduals of P[ `racile having an average of 3[8 GSCs In this study we investigated by GC!MS the vol! per leaf had only 3[2) of their leaves damaged by atiles emitted from the aerial parts of P[ `racile and P[ herbivores\ mostly generalist grasshoppers\ while ruderale and con_rmed their identity[ The hypothesis plants with a mean value of 1[4 GSCs per leaf su}ered that these volatiles exert a synergistic e}ect on the a _ve!fold higher rate of herbivory[ The same study insecticidal properties of a!terthienyl\ a phototoxic polyacetylenic derivative also present in P[ `racile and P[ ruderale\ was also tested against larvae of the Euro! pean corn borer\ Ostrinia nubilalis "HuÃbner# "Lep! Author to whom correspondence should be addressed idoptera] Pyralidae#[ Although O[ nubilalis is not a

312 313 GABRIEL GUILLET et al[

Table 0[ Chemical constituents in the leaves of Porophyllum ruderale according to di}erent sampling techniques[

Sampling technique

Direct $ Steam distillation Head!space ") of the integrated ") of the integrated ") of the integrated Chemical constituent FID trace# FID trace# FID trace#

Monoterpenes a!pinene n[d[ n[d[ n[d[ sabinene 04 02 11 1\2!dihydro!0\7!cineole n[d[ 1 n[d[ myrcene 5 4 6 limonene 603 621 778 trans!b!ocimene 0 n[d[ n[d[ terpinolene n[d[ n[d[ n[d[ Sesquiterpene b!cubebene n[d[ n[d[ n[d[ Fatty acid derivatives 6!tetradecene n[d[ 09 n[d[ cis!3!decenal n[d[ 6 n[d[ pentadecanal 05 19 n[d[ heptadecanal 129 1 n[d[

The abbreviation n[d[ refers to not detected[ $The extract was obtained from a direct sampling of the liquid secreted from the glandular secretory cavities present on the leaves[

pest of the genus Porophyllum\ this insect has been steam distillation "Table 1#[ Di}erent fatty acid reported to feed on over 199 herbaceous plants includ! derivatives\ 6!tetradecene\ cis!3!decenal\ pentadecanal ing many Asteraceae ð02Ł[ Given the frequent occur! and heptadecanal\ were also detected in the extracts rence of monoterpene ð03Ł and thiophene ð04Ł deriva! obtained by the steam distillation of leaves\ which tives in the Asteraceae family\ Ostrinia nubilalis is contributed 2[8) and 4[2) of the total extracts in P[ therefore likely to encounter in its natural host plants ruderale and P[ `racile respectively "Tables 0 and 1#[ these two types of phytochemicals[ A sesquiterpene\ b!cubebene\ was also identi_ed in the steam distillation extract of P[ `racile "Table 1#[ Chemical analysis of the liquid sampled directly RESULTS from the GSCs in the leaves of P[ ruderale revealed a similar pro_le of monoterpenes to the one obtained Chemical Analysis with the head!space technique except that a small Volatiles that were collected from P[ `racile and P[ amount of trans!b!ocimene "9[0)# was found "Table ruderale using the static head!space technique were 0#[ The relative amount of the fatty acid derivatives in mostly monoterpenes[ Sabinene\ myrcene and lim! P[ ruderale was 13[5) of the integrated FID trace in onene constituted 80[7) of the integrated FID trace the direct sampling compared to 2[8) and unde! of chemicals emitted from the leaves of P[ ruderale tectable levels in the steam distillation and head!space and a!pinene\ sabinene and myrcene accounted for techniques respectively "Table 0#[ 72[8) of the volatile substances collected from the One substance\ 6!tetradecene\ constituted 66[9) leaves of P[ `racile "Tables 0 and 1#[ and 89[1) of the integrated FID trace obtained from The essential oil obtained by steam distillation of the stems of P[ ruderale by steam distillation and head! leaves provided a similar pro_le of monoterpenes to space samplings respectively "Table 2#[ Monoterpenes the results obtained with the head!space technique for were not detected in the stems of P[ ruderale according P[ ruderale except that a small amount of 1\2!dihydro! to head!space sampling\ although limonene con! 0\7!cineole\ 9[1) of the integrated FID trace\ was stituted 7[5) of the integrated FID trace of the extract also detected "Table 0#[ As for P[ `racile\ the same collected by steam distillation "Table 2#[ monoterpenes were identi_ed by both sampling tech! niques\ but the relative proportions di}ered[ This was Insect Toxicity especially evident for a!pinene and sabinene\ which represented 06[6) and 2[4) respectively of the The relative growth rate of O[ nubilalis larvae extract obtained with the head!space sampling and exposed to volatiles emitted from the foliar GSCs of 1[2) and 19[4) of the extract obtained through P[ ruderale was not signi_cantly di}erent from the Monoterpenes from Porophyllum spp[ 314

Table 1[ Chemical constituents in the leaves of Porophyllum gracile according to di}erent sampling techniques[

Sampling technique

Steam distillation Head!space ") of the integrated ") of the integrated Chemical constituent FID trace# FID trace#

Monoterpenes a!pinene 1[2 06[6 sabinene 19[4 2[4 1\2!dihydro!0\7!cineole n[d[ n[d[ myrcene 39[5 51[6 limonene n[d[ n[d[ trans!b!ocimene n[d[ n[d[ terpinolene n[d[ n[d[ Sesquiterpene b!cubebene 8[0 n[d[ Fatty acid derivatives 6!tetradecene 0[3 n[d[ cis!3!decenal n[d[ n[d[ pentadecanal 1[8 n[d[ heptadecanal 0 n[d[

The abbreviation n[d[ refers to not detected[

Table 2[ Chemical constituents in the stems of Porophyllum ruderale "Jacq[# Cass[ var[ macrocephalum "DC[# according to di}erent sampling techniques[

Sampling technique

Steam distillation Head!space ") of the integrated ") of the integrated Chemical constituent FID trace# FID trace#

Monoterpenes a!pinene n[d[ n[d[ sabinene n[d[ n[d[ 1\2!dihydro!0\7!cineole n[d[ n[d[ myrcene n[d[ n[d[ limonene 7[5 n[d[ trans!b!ocimene n[d[ n[d[ terpinolene n[d[ n[d[

Sesquiterpene b!cubebene n[d[ n[d[

Fatty acid derivatives 6!tetradecene 66 89[1 cis!3!decenal n[d[ n[d[ pentadecanal n[d[ n[d[ heptadecanal n[d[ n[d[ b!cubebene n[d[ n[d[

The abbreviation n[d[ refers to not detected[

control treatment "Fig[ 0#[ The relative growth rate of relative growth rate mediated by a!terthienyl was O[ nubilalis larvae was\ however\ reduced by 06) ampli_ed 1[3!fold\ i[e[ 30) vs 06)\ when insects were when a!terthienyl was added in a meridic diet at a concurrently exposed to the volatiles released from concentration of 49 ppm[ The reduction of the larval the GSCs of the leaves of P[ ruderale "Fig[ 0#[ A nearly 315 GABRIEL GUILLET et al[

DISCUSSION The present study suggests that the volatile mono! terpenes released from the leaves of P[ ruderale are mainly sequestered in the GSCs[ There was indeed no monoterpene detected in the stems of P[ ruderale using static head!space sampling\ although a small level of limonene was found with the steam distillation extrac! tion[ A similar pro_le of monoterpenes was fur! thermore observed in the extracts obtained by either the head!space or the steam distillation techniques using whole leaves\ and the direct sampling of the liquid directly secreted from the GSCs "Table 0#[ It is expected that a sequestration of volatile monoterpenes in the GSCs also occurs for P[ `racile*a typical pung! ent smell was released when the GSCs of both P[ `racile and P[ ruderale were squeezed*\ although the Fig[ 0[ Synergistic e}ects of the volatiles released from the content was not measured[ The sequestration of glandular secretory cavities of the leaves of Porophyllum monoterpenes into storage organs is well established ruderale "Asteraceae# and a!terthienyl on the relative growth ð05Ł[ For example\ a mixture of monoterpenes includ! rate of third instar Ostrinia nubilalis larvae "the P value for ing among others sabinene\ camphor and a!pinene is the interaction between volatiles and a!terthienyl was 9[941#[ Error bars represent the standard deviation and treatments contained in the trichomes of Mentha piperita and associated with distinct letters di}er signi_cantly "Tukey Cleome spinosa ð06\ 07Ł[ Indole and monoterpenes are HSD multiple comparisons\ P³9[94#[ also sequestered in GSCs in leaves of Ta`etes erecta ð08Ł[ It is likely that the monoterpenes\ which constitute the major part of the chemicals emitted from the GSCs of P[ ruderale "Tables 0 and 1#\ are responsible for the repellent activity previously reported for P[ `racile and P[ ruderale against red!legged grasshopper adults ð4Ł[ Although our results showed that the volatile monoterpenes released from the GSCs of the leaves of P[ ruderale did not reduce the relative growth rate of European corn borer larvae\ these monoterpenes synergized the e}ects of a!terthienyl against the same insect "Fig[ 0#[ This synergism between volatile mono! terpenes and a!terthienyl in the reduction of the rela! tive growth rate of O[ nubilalis was apparently due to a nearly two!fold increase in the concentration of a! terthienyl in larvae when they were exposed to the volatiles released from the foliar GSCs of P[ ruderale "Fig[ 1#[ Other monoterpenes have been demonstrated to enhance the uptake of both hydrophilic and lipophilic Fig[ 1[ E}ects of the volatile monoterpenes released from the chemicals into organic structures[ For example\ Yam! glandular secretory cavities of the leaves of Porophyllum ane et al[ ð19Ł showed that limonene and cineole ruderale "Asteraceae# on the retention of a!terthienyl in _fth enhance by 2[5! and 84!fold respectively the par! instar Ostrinia nubilalis larvae fed with a meridic diet con! titionning of a hydrophilic drug\ 4!~uorouracil\ from taining a!terthienyl[ Error bars represent the standard aqueous solutions into human stratum corneum[ deviation and treatments associated with distinct letters di}er Ogiso et al[ ð10Ł observed that cineole had a similar signi_cantly "Tukey HSD multiple comparisons\ P³9[94#[ e}ect on the transdermal penetration of a lipophilic drug\ indomethacin\ in rat[ Monti et al[ ð11Ł also used di}erent terpenes as penetration enhancers of a chem! otherapeutic drug\ dapiprazole\ through hairless mouse skin[ The increase of the chemical uptake into organic two!fold increase in the retention of a!terthienyl in tissues mediated by some monoterpenes appears to be larval tissues of the European corn borer was also related to an alteration of the lipid organization in observed in presence of the volatiles released from the cellular membranes or in intercellular lamellar struc! foliar GSCs of P[ ruderale "Fig[ 1#[ tures ð19Ł[ In sophisticated studies involving comp! Monoterpenes from Porophyllum spp[ 316 lementary results obtained from di}erential scanning constituents with those obtained with volatilization calorimetry\ small!angle X!ray di}raction and techniques as above[ Direct sampling was not enhancer uptake in human stratum corneum\ attempted with the foliar glands of P[ `racile given Cornwell et al[ ð12Ł and Yamane et al[ ð13Ł have shown their smaller dimensions[ that some monoterpenes\ including cineole and limon! Chemical analysis[ GC analysis was performed with ene\ partially disrupt the lipid bilayer organisation of a FID and the columns used were a fused silica Dur! cellular membranes[ abond DB!4 "9[14 mm _lm thickness\ 29 m×9[14 mm An alternative explanation for the higher retention i[d[#\ a fused silica Durabond DB!0 "0[9 mm _lm thick! of a!T in O[ nubilalis larvae exposed to volatiles "Fig[ ness\ 29 m×9[14 mm i[d#\ and a fused silica Durabond 1# is an inhibitory e}ect that these volatiles may exert DB!Wax "9[14 mm _lm thickness\ 29 m×9[14 mm i[d[# on the metabolic detoxi_cation of a!T which relies on "J + W Scienti_c\ Folsom\ CA#[ For all analyses\ the cytochrome P!349 enzymatic complex in this insect the temperature program was as follows] 39> initial ð14Ł[ In support of this hypothesis\ it has been estab! temperature\ 1>:min to 149> with helium as the carrier lished that b!myrcene and d!limonene inhibit the in gas[ The injector and detector temperatures were 129> vitro metabolic activity of a cytochrome P!349 and 149>\ respectively[ Injections of 0 ml were made in monooxygenase "CYP1B0# in rat liver microsomes the splitless mode and changed to the split mode after ð15Ł[ 9[5 min[ Retention indices were calculated relative to Overall\ results of the present study indicated that n!alkane standards[ Electron impact MS "ca 099 ng the co!occurrence of monoterpenes with a!terthienyl sample# were obtained using an ionizing potential of may represent an integrated defensive strategy used 69 eV[ by Porophyllum spp[ to improve\ by synergism\ their Insect toxicity[ To verify if a!terthienyl and volatile chemical protection against herbivorous insects[ A monoterpenes\ which co!occur in P[ ruderale and P[ {solvent hypothesis| has already been proposed that `racile\ exert additive toxic "insecticidal# e}ects suggests monoterpenes may serve as agents for storage against the European corn borer\ Ostrinia nubilalis and delivery of lipophilic secondary compounds to an HuÃbner "Lepidoptera] Pyralidae#\ a simple factorial attacked site of host plants ð16Ł However\ the present experiment was designed[ The following treatments study suggests that monoterpenes may also enhance were performed] non!exposed larvae "control#\ larvae the retention of toxic secondary compounds in insect exposed to either volatile monoterpenes or a!terthi! tissues[ Given the frequent co!occurrence in plants enyl\ and larvae simultaneously exposed to both of monoterpenes and toxic higher molecular weight chemicals[ compounds and assuming that a similar synergism to Newly molted third instar O[ nubilalis larvae the one observed in the present study also occurs obtained from a laboratory colony were maintained between monoterpenes and other classes of secondary and fed a meridic diet as described in ð18Ł[ A meridic compounds\ it is expected that monoterpenes may diet was warmed to 24> before adding a!terthienyl at represent a widespread component of chemical a concentration of 49 ppm via a Me1CO soln added at defences[ a rate of 4 ml:g[ This concentration of 49 ppm of a! terthienyl is representative of the amount of thiophene derivatives occurring in tissues of Porophyllum spp EXPERIMENTAL ð6Ł[ and other Asteraceae ð6\ 29Ð21Ł[ An equivalent

Plants[ Seeds of P[ ruderale were provided by Xavier amount of Me1CO was added in the diet for treat! Lozoya from Xochitepec "Mexico# while those of P[ ments without a!terthienyl[ Diets were then incubated `racile were collected by Guillet in the area of Tucson in darkness at room temperature for two hr to let agar

"\ USA# in August 0883[ Seeds of both species solidify and Me1CO evaporate[ Exposure of insects to were germinated in water and then transplanted in volatile monoterpenes was controlled by inserting a vermiculite[ Plants were fertilized weekly with a Hoag! leaf of P[ ruderale harboring 4Ð6 GSCs of 1Ð2 mm of land|s solution ð17Ł and grown under greenhouse con! length inside a glass petri dish in which larvae were ditions at 19>:7 hr night and 14>:05 hr day[ Natural fed[ Leaves were individually inserted under a _ne lighting supplemented with high!pressure sodium metallic mesh of 14 mm of diameter that was glued on lamps provided a daytime irradiance of 299249 the inside of petri lids[ This protocol made the leaves mmol m−1 s−0 photosynthetic photon ~ux density[ inaccessible to O[ nubilalis larvae\ although it did not Chemical samplin`[ The techniques used to extract prevent the volatiles released from GSCs from reach! the volatile compounds from plant tissues\ steam dis! ing the larvae[ For treatments with exposure to tillation and static head!space techniques have been volatiles\ the GSCs were physically crushed at the described previously ð26\ 27Ł[ The steam distillation beginning of the experiment whereas the GSCs were technique provides the full spectrum of volatiles in gently dissected out of leaves with a razor blade and the tissue\ although some rearrangements may occur\ discarded in treatments with no exposure to volatiles[ while the static head space technique yield the more Leaves from each dish were replaced every day[ At readily volatalized materials[ Direct sampling by mic! the beginning of the experiment\ 04 third instar larvae ropipette of the liquid contained in the GSCs of the were weighed together then transferred in a glass petri leaves of P[ ruderale was also undertaken to compare dish containing _ve cubes of diet\ ca 9[8 cm2 each\ with 317 GABRIEL GUILLET et al[ or without a!terthienyl according to the respective [ McGraw!Hill Book Company\ New York\ treatment[ For each of the four treatments\ ten rep! 0855[ licates were performed and insects were exposed to 2[ Munz\ P[ A[ and Keck\ D[ D[\ A ~ora[ near!UV light as described in ð22Ł to stimulate the University of California press\ Berkeley\ 0862[ photosensitization mediated by a!terthienyl ð23Ł[ The 3[ Monteiro\ W[ R[\ Castro\ M[ D[\ Fahn\ A[ and experiment lasted 37 hr and the _nal wt of insects Caldeira\ W[\ Nord[ J[ Bot[\ 0884\ 04\ 58[ was measured to determine the relative growth rate 4[ Guillet\ G[\ Lorenzetti\ F[\ Belanger\ A[\ Arnason\ "weight gain:initial weight:day#[ The potential insec! J[ T[ and Bernays\ E[ A[\ J[ Ecol[\ 0886\ 74\ 536[ ticidal activity was assumed to be higher in treatments 5[ Lozoya\ M[ M[ and Gaspar\ I[\ Fitoterapia\ 0882\ with lower growth rate ð24Ł[ 53\ 24[ In a second experiment\ the e}ects of the volatiles 6[ Downum\ K[ R[\ Keil\ D[ J[ and Rodriguez\ E[\ released from the foliar GSCs of P[ ruderale on the Biochem[ Syst[ Ecol\ 0874\ 02\ 098[ retention of a!terthienyl in O[ nubilalis larvae were 7[ Bohlmann\ F[\ Jakupovic\ J[\ Robinson\ H[ and investigated[ Actively feeding mid!_fth instar larvae King\ R[ M[\ Phytochemistry\ 0879\ 08\ 1659[ were exposed to the volatiles released from the GSCs 8[ Bohlmann\ F[ and Zdero\ C[\ Phytochemistry\ of a leaf of P[ ruderale as above while insects were fed 0868\ 07\ 230[ for 13 hr in darkness on a meridic diet containing a! 09[ Bohlmann\ F[\ Zdero\ C[\ King\ R[ M[ and Robin! terthienyl at a concentration of 49 ppm[ The per! son\ H[\ Phytochemistry\ 0872\ 11\ 0924[ itrophic membrane of larvae was then removed before 00[ Chan\ G[ F[ Q[\ Lee\ M[ M[\ Glushka\ J[ and determining their a!terthienyl content to reduce vari! Towers\ G[ H[ N[\ Phytochemistry\ 0868\ 07\ 0455[ ations related to diet remains possibly occurring in 01[ Downum\ K[ R[ and Towers\ G[ H[ N[\ J[ Nat[ gut lumen of insects[ This was done by cutting the Prod[ "Lloydia#\ 0872\ 35\ 87[ head and the two or three last abdominal segments of 02[ Hudon\ M[\ LeRoux\ E[ J[ and Harcourt\ D[ G[ in each larva so that the peritrophic membrane and its Biolo`y and Population Dynamics of Invertebrate content could be pulled out using _ne dissection Crop Pests\ ed[ G[E[ Russell[ Intercept Ltd[\ And! tweezers[ Insect tissues without the peritrophic mem! over\ Hampshire\ U[K[\ 0878\ p[ 0[ brane were dipped in 09 ml of hexane and agitated for 03[ Gershenzon\ J[ and Croteau\ R[\ In Herbivores] 37 hr in darkness to solubilize a!terthienyl[ An aliquot Their Interactions with Secondary Metabolites\ of 0[4 ml of the hexane in which larvae were placed was Vol[ 0\ Academic Press\ New York\ p[ 054[ then centrifuged "05 999 g\ 4 min# and the supernatant 04[ Bohlmann\ F[\ Burkhardt\ T[ and Zdero\ C[\ Nat! was kept for HPLC analysis[ The yield for the extrac! urally occurrin` acetylenes[ Academic Press\ tion of a!terthienyl according to this procedure was London\ 0862[ shown to be higher than 89) in a preliminary experi! 05[ Gershenzon\ J[\ J[ Chem[ Ecol[\ 0883\ 19\ 0170[ ment[ Insect tissues were then dried and weighed to 06[ Amelunxen\ F[ and Arbeiter\ H[\ Z[ P~an! determine the concentration of a!terthienyl in a dry zenphysiol\ 0858\ 50\ 62[ wt basis[ Ten O[ nubilalis larvae were used per replicate 07[ Amelunxen\ T[\ Wahlig\ T[ and Arbeiter\ H[\ Z[ and six replicates were performed for each treatment[ P~anzenphysiol\ 0858\ 50\ 57[ a!terthienyl analysis[ Concentration of a!terthienyl 08[ Russin\ W[ A[\ Uchytel\ T[ F[\ Feistner\ G[ and was determined by HPLC using a Beckman Gold Durbin\ R[ D[\ Amer[ J[ Bot[\ 0877\ 64\ 0676[ System equipped with a solvent module "model 015#\ 19[ Yamane\ M[ A[\ Williams\ A[ C[ and Barry\ B[ a UV! detector "model 057# set to 249 nm and an W[\ Int[ J[ Pharm[\ 0884\ 005\ 126[ autosampler "model 491#[ A C!07 reverse phase col! 10[ Ogiso\ T[ and Iwaki\ M[\ Paku\ T[\ J[ Pharm[ Sc[\ umn "Beckman\ 4 ODS\ 14 cm 3[5 mm# was used with 0884\ 73\ 371[ acetonitrile]water "2]0# at a ~ow rate of 0[9 ml:min[ 11[ Monti\ D[\ Saettone\ M[ F[\ Giannaccini\ B[ and Under these conditions\ a!terthienyl had a retention Galli Angeli\ D[\ J[ Controlled Release\ 0884\ 22\ time of 05[2 min and its concentration was determined 60[ by comparing the peak areas of samples to those of a 12[ Cornwell\ P[ A[\ Barry\ B[ W[\ Bouwstra\ J[ A[ standard curve made with pure a!terthienyl syn! and Gooris\ G[ S[\ Int[ J[ Pharm[ "Amst[#\ 0885\ thesized as described in ð25Ł[ 016\8[ 13[ Yamane\ M[ A[\ Williams\ A[ C[ and Barry\ B[ Acknowled`ements*This work was funded by W[\ J[ Pharm[ Pharmacol[\ 0884\ 36\ 867[ NSERC and FCAR postgraduate scholarships to GG 14[ Iyengar\ S[\ Arnason\ J[ T[\ PhilogeÁne\ B[ J[ R[\ and and an NSERC strategic grant to JTA[ Morand\ P[ and Westiuk\ N[ H[\ Pestic[ Biochem[ Physiol[\ 0889\ 26\ 043[ 15[ De Oliveira\ A[ C[\ Ribeiro Pinto\ L[ F[ and REFERENCES Paumgartten\ J[ R[\ Toxicol[ Letters\ 0886\ 81\ 28[ 0[ Rickett\ H[ W[\ Wild Flowers of the United States\ 16[ Lewinsohn\ E[\ Gijzen\ M[\ Muzika\ R[ M[\ Vol[ 2] The southwestern states[ McGraw!Hill Burton\ K[ and Croteau\ R[\ Pl[ Physiol[\ 0882\ Book Company\ New York\ 0855[ 090\ 0910[ 1[ Rickett\ H[ W[\ Wild Flowers of the United States\ 17[ Hoagland\ D[ R[ and Arnon\ D[ I[\ University Monoterpenes from Porophyllum spp[ 318

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