Phytochemistry\ Vol[ 37\ No[ 5\ pp[ 820Ð828\ 0887Þ 0887 Elsevier Science Ltd[ All rights reserved\ Pergamon Printed in Great Britain

9920Ð8311:87 ,08[99¦9[99PII] S9920Ð8311"86#90992Ð9

ACYLPHLOROGLUCINOLS AND FLAVONOID AGLYCONESPRODUCED BY EXTERNAL GLANDS ON THE LEAVES OF TWO

DRYOPTERIS FERNS AND CURRANIA ROBERTIANA

ECKHARD WOLLENWEBER\ JAN F[ STEVENS\�% MONIKA IVANIC$ and MAX L[ DEINZER%

Institut fu�r Botanik der Technischen Universita�t\ Schnittspahnstrasse 2\ D!53176 Darmstadt\ Germany^ % Dept[ ofChemistry^ $ Dept[ of Biochemistry and Biophysics\ ALS Building 0996\ Oregon State University\ Corvallis\

OR 86220\ U[S[A[

"Received 11 September 0886#

Key Word Index*Dryopteris villarii ^ D[ ar`uta^ Currania robertiana^ Pteridaceae^ ferns\ leafexudates^ acylphloroglucinols^ ultrastructure of glandular trichomes[

Abstract*The sparse resinous leaf exudates of Dryopteris villarii and D[ ar`uta contain acylphloroglucinolsthat have previously only been found in the rhizomes of Dryopteris species[ The phloroglucinol derivativeswere characterized as representatives of the para!aspidin\ desaspidin and albaspidin series by 1!D NMRspectroscopy and atmospheric pressure chemical ionization mass spectrometry "APCI!MS!MS#[ Mass frag!mentation proved to be of diagnostic value to identify the acyl side chains and also allowed distinction betweendesaspidins and isomeric albaspidin analogues[ The frond exudate of Currania robertiana was devoid ofacylphloroglucinols\ but was found to contain ~avonoid aglycones[ The acylphloroglucinols and ~avonoidaglycones are presumably produced by external glands on the leaves\ the presence and ultrastructure of whichwas examined by scanning electron microscopy[ Þ 0887 Elsevier Science Ltd[ All rights reserved

INTRODUCTION

Extensive studies on the chemical composition offrond exudates from Gymnogrammoid ferns "generaCheilanthes\ Notholaena\ Pityro`ramma and someother Pteridaceae# have revealed the presence of a richarray of ~avonoid aglycones ð0Ð2Ł\ diterpenoids andtriterpenoids ð3Ð5Ł[ All fern species previously exam!ined for exudate ~avonoids or other phenolics ð6Łoriginate from both Americas\ from Asia\ and fromAustralia "Platyzoma ð7Ł#[ This is the _rst study ofthe foliar exudates of two European Alpine ferns\Currania robertiana "Ho}m[# Wherry ðSyn[] Gym!nocarpium robertianum "Ho}m[# Newm[^ Dryopterisrobertiana "Ho}m[# C[ Chr[Ł and Dryopteris villarii"Bell[# Woynar[

Preliminary TLC analysis of the frond exudates ofD[ villarii and D[ ar`uta "Kaulf[# Watt[ from Cal!ifornia indicated the presence of non!~avonoid phe!nolics which were subsequently isolated and identi_edas acylphloroglucinols by spectroscopic techniques[Although acylphloroglucinols are well!known asinternal gland products from rhizomes of many ferns\in particular Dryopteris ð8\ 09Ł\ their presence in leaf

� Author to whom correspondence should be addressed[

820

exudates produced by external glands is reported herefor the _rst time[

RESULTS AND DISCUSSION

Identi_cation of leaf exudate constituents

In the following\ Penttila� and Sundman|s systemfor assigning names to acylphloroglucinols ð00Ð02Łand numbering of atoms as in Ref[ ð03Ł is used[ Thefrond exudate of D[ ar`uta yielded para!aspidin AA"0!AA# and four acylphloroglucinols of the desaspidinseries "1!AP\ 1!PP\ 1!AB\ and 1!PB# by column chro!matography on polyamide and semi!preparativeHPLC on RP!07[ In addition\ six acylphloroglucinolswere isolated from the frond exudate of D[ villariiwhich were identi_ed as the albaspidin homologues 2!

AP\ 2!PP\ 2!AB\ 2!PB\ 2!BB and aspidinol "3#[ Struc!ture assignment was achieved by a combination ofatmospheric pressure chemical ionization mass spec!trometry "APCI!MS# and 1!D NMR spectroscopy[By co!TLC with markers\ the frond exudate of C[robertiana was found to contain the ~avones\ apig!enin\ apigenin!6!methylether\ apigenin!3?!methy!lether\ apigenin!6\3?!dimethylether and the ~avonols\kaempferol\ kaempferol!2!methylether\ kaempferol!3?!methylether\ kaempferol!2\3?!dimethylether\

ECKHARD WOLLENWEBER et al[821

kaempferol!2\6\3?!trimethylether and herbacetin!2\7\3?!trimethylether[ The latter ~avonol is a ratherrare compound which\ to our knowledge\ was pre!viously found only _ve times from natural sources\including a fern Pityro`ramma trian`ularis var[ tri!an`ularis "now Penta`ramma# ð04Ł[ The foliar exu!dates of D[ villarii and D[ ar`uta were found to bedevoid of ~avonoids[

Compound 1!AB "Mr 307\ C11H15O7# was obtainedas the major constituent of the frond exudate of D[ar`uta[ Its 0H NMR spectrum showed characteristicsignals for an acylphloroglunicol[ Four major low!_eld resonances attributable to "hydrogen!bonded#hydroxyls "dH 8[87\ 00[20\ 05[28 and 07[39#\accompanied by some four minor signals due to keto!enol tautomerism[ The methylene bridge linking thetwo ring systems appeared as a broad singlet at dH2[42which interacted with C!0 and C!0? of both rings inthe HMBC spectrum[ The H!4? singlet at dH5[94 wasassigned to an aromatic proton\ indicating that onering system was aromatic in nature[ The other ringwas identi_ed as a _licinic acid unit with a `em!dime!thyl group resonating at dH0[44[ This signal coupledwith the 02C resonance at dC13[7 in the HMQC spec!trum of 1!AB[ The substitution pattern of the aro!matic "phloroglucinol# ring was determined to be 2?!acyl\ 4?!H\ 5?!OMe[ The aromatic proton signalshowed interactions with all ring carbons except C!1?

bearing a free hydroxyl with dH 05[28\ which excludesthe possibility of 1!AB being an iso!desaspidin hom!ologue[ Furthermore\ a methoxy group at C!5? fol!lowed from the observation that the free hydroxylortho to C!4? interacted with C!2?\ C!4? and with thecarbon to which the hydroxyl is attached\ C!3?[ The0H NMR spectrum further demonstrated the presenceof an acetyl and a butyryl side chain[ Table 0 lists all0H and 02C shifts for 1!AB assigned on the basis of0HÐ02C couplings observed in the HMQC and HMBCspectra as well as by comparison with NMR datareported for desaspidin BB ð03Ł[ The acetyl functionwas located on the _licinic acid unit by mass spec!trometry "see below#[ Compound 1!AB was thus ident!i_ed as desaspidin AB[

The accompanying acylphloroglunicols from D[ar`uta were identi_ed by 0H NMR and mass spec!trometry only[ Compounds 1!AA\ 1!AP and 1!PB

showed 0H NMR spectra similar to that of 1!AB\except for the acyl side chain signals\ and were there!fore identi_ed as desaspidin homologues[ Compound0!AA "Mr 289\ C19H11O7# lacked the aromatic H!4?signal but showed a C!methyl at dH 1[03 instead[ The0H NMR spectrum of 0!AA further showed two acetylmethyls[ The structure of 0!AA was assigned as para!aspidin AA and con_rmed by mass spectrometry[

The Mr of compound 2!BB "Mr 359\ C14H21O7# sug!gested that 2!BB was identical with or closely related

Leaf surface acylphloroglucinols from Dryopteris 822

Table 0[ 0H and 02C NMR data for phloroglucides 1!AB\ 2!PB and 2!BB

Atom No[ 1!AB dH 1!AB dC 2!PB dH 2!PB dC 2!BB dH 2!BB dC

0 097[54 009[76 009[771 076[52 076[65 076[662 000[28 097[10 097[103 088[93 088[17 088[174 33[37 33[41 33[415 061[98 062[30 062[316 2[42 br s 05[70 2[20 s 07[07 2[20 s 07[127 192[60 195[41 195[428 1[62 s 18[43 2[05 m 32[02 2[04 m 32[02

09 0[06 t "6[1#$ 07[12 0[58 m 07[1200 0[90 t "6[3#¦ 03[0601 0[44 s 13[66 0[440 s� 14[40� 0[43� 14[49�02 0[44 s 13[66 0[37 s� 13[27� 0[37 s� 13[27�0? 093[68 009[74 009[771? 052[30 076[79 076[662? 095[48 097[03 097[103? 053[19 087[56 088[174? 5[94 s 82[01 33[39 33[415? 051[26 062[30 062[317? 195[51 196[33 195[428? 1[88 m 34[80 2[12 m 24[97 2[04 m 32[02

09? 0[69 m 07[26 0[58 m 7[48 0[58 m 07[1200? 9[88 t "6[3#$ 03[14 0[90 t "6[2#$ 03[06 0[90 t "6[3#$ 03[0601? 0[434 s� 14[37� 0[43 s� 14[49�02? 0[36 s� 13[27 0[37 s� 13[27�3!OH 07[39 s 07[53 s 07[53 s5!OH 8[87 s 01[24 s 01[24 s1?!OH 05[28 s3?!OH 00[20 s 07[46 s 07[53 s5?!OH 01[24 s 01[24 s5?!OMe 2[74 s 44[71

� Assignments interchangeable[$ "J in Hz#[

to the known fern phloroglucinol\ aspidin BB[However\ this possibility was immediately ruled outbecause of the absence of O!methyl resonances in the0H NMR spectrum[ In the 02C NMR spectrum\ therewere only 01 carbon signals indicating a symmetricalmolecule consisting of two butyryl_licinic acids linkedthrough a methylene bridge[ Unambiguous assign!ment of the 0H and 02C resonances followed from 0HÐ02C HMQC and HMBC spectroscopy "Table 0#[ Thestructure of 2!BB being identical with albaspidin BBwas con_rmed by comparison of our NMR data withthose reported for a number of acylphloroglucinolsð03Ł[ In the HMBC spectrum\ the `em!dimethylgroups did not interact with C1O carbon of the _l!icinic acid rings[ This indicates that the preferred solu!tion con_rmation is the tautomer with the `em!dime!thyl para to the keto function[ From the HMQC spec!trum it became clear why only 01 signals wereobserved and not the expected 02 signals] it appearedthat carbons 6\ 09 and 09? all resonated at dc 07[12\since there were two cross peaks for this signal\ onewith H!6 at dH 2[20 and the other with the multiplet at

dH 0[58 assigned to H!09:09? by HMBC correlations"Table 0#[

Compound 2!PB "Mr 335\ C13H29O7# was identi_edas albaspidin PB in a similar fashion[ Due to thenear!symmetry of the molecule\ double signals wereobserved for the ring carbons of both _licinic acidunits[ The carbons resonating at the same frequenciesas the ring carbons in albaspidin BB were identi_edas those belonging to the butyryl_licinic acid unit[This piece of information could not be inferred fromHBMC correlations because there were no inter!actions between nuclei across the acyl C1Os[ Assign!ment of the butyryl and propionyl signals readily fol!lowed from the HMBC spectrum[ The acyl carbon atdC 195[4 showed cross peaks with two methylene pro!ton signals "dH 2[05 and 0[58# and the terminal methylprotons at dH 0[90\ which were therefore attributed tothe butyryl function[ Likewise\ within the propionylside chain\ acyl C!7? interacted with the methyleneprotons at dH 2[12 "H!8?# and the methyl protons atdH 0[06 "H!09?#[ Full assignment of signals is given inTable 0[

ECKHARD WOLLENWEBER et al[823

Fig[ 0[ HPLC!APCI!MS analysis of crude phloroglucinol mixtures of D[ ar`uta "upper panels# and D[ villarii "lower panels#[Total ion current "TIC# chromatograms were recorded in the positive ion mode from m:z 059 to m:z 459 "left#[ Theacylphloroglucinols yielded predominantly pseudo!molecular ðMHŁ¦ ions "middle# which were collisionally activated to give

X and Y cleavage ions in the MS!MS mode "CAD spectra\ right#[

The accompanying minor acylphloroglucinols\ 2!

AP\ 2!AB\ and 2!PP\ were obtained from the frondexudate of D[ villarii in very small amounts[ Theirstructures were tentatively assigned as the albaspidinhomologues AP\ PP and AB by mass spectrometry[

Compound 3 "Mr 113\ C01H05O3# was identi_ed asaspidinol B by 0H\ 02C\ 0HÐ02C HMQC and HMBCspectroscopy[ The butyryl function was located parato the methoxy substituent\ because the aromatic pro!ton "H!4?\ at dH 4[85# correlated with all ring carbonsbut C!1?[ This indicates that the aromatic proton ispara to C!1? and meta to C!0? carrying the butyryl sidechain[

Mass spectrometry

The composition of the crude phloroglucinol mix!tures of D[ ar`uta and D[ villarii was examined byHPLC!APCI!MS in the single and tandem MS modes"Fig[ 0#[ The acylphloroglucinols yielded intense pro!tonated molecular ðMHŁ¦ ions without signi_cantfragmentation in the single MS mode[ Fragmentationof the ðMHŁ¦ ions was induced by collisions with Argas molecules in the tandem!MS mode[ Basically\ twofragmentation pathways were discerned\ "0# cleavageof the methylene bridge on either side of C!6\ and "1#loss of the acyl side chain ð05Ł[ The _rst route yieldedthe most intense fragment ions which proved to be ofgreat diagnostic value in the identi_cation of bothacyl residues "Table 1\ Fig[ 1#[ In the albaspidins\ thecharge is preferentially retained on the _licinic acidfragments\ XH¦ and YH¦\ after cleavage of the meth!ylene bridge[ By contrast\ para!aspidin!AA and thedesaspidins yielded predominantly Y?H¦ and YH¦

ions as inferred from homologues whose XH¦ and

YH¦ fragments di}er in mass[ Hence it seems reason!able to assume that the contribution of the YH¦ ionsto the ion current is signi_cantly larger than the con!tribution of XH¦ ions for all desaspidins[ This indi!cates that the charge is retained on the phloroglucinolunit rather than on the _licinic acid unit "cf ion inten!sities in Table 1#[ Clearly\ charge retention is favouredon the least acidic oxygen substituents\ that is\ onthe phloroglucinol hydroxyl because the _licinic acidoxygen substituents participate in tautomeric proton!exchange processes[ As can be seen from Table 1\di}erences in the ion intensities of the Y?H¦ ions allowdistinction between desaspidins and their isomericalbaspidins[ Consider\ for instance\ the two isomericacylphloroglucinols\ 1!AB and 2!AP\ whose X cleav!age products are of equal mass "and Y products ofequal mass#[ They di}er signi_cantly in the intensitiesof the Y?H¦ ions] 75) observed for 1!AB and only01) for 2!AP "Fig[ 0\ Table 1#[

In the second pathway\ fragment ions are formedby loss of an acyl neutral\ O1C1CHR\ from theðMHŁ¦ ion which are of additional diagnostic valuein the albaspidins[ Loss of the acyl substituent fromboth XH¦ and YH¦ yields protonated _licinic acidwith m:z 044[ Although the desaspidins contain anacyl_licinic acid unit\ the m:z 044 ion was onlyobserved in spectra of the albaspidins "ðX−O1C!1CHR\ usually× 09) of the base peak#\ and thusprovides another diagnostic ion to distinguishbetween desaspidins and albaspidins[ Additional frag!ment ions may arise as a result of loss of water fromðMHŁ¦\ XH¦ and YH¦ ions[

Acylphloroglucinols have previously been analysedby other {soft| ionization techniques\ i[e[ CI!MS\ FI!MS\ FD!MS and FAB!mass "cf ð06Ł and refs cited

Leaf surface acylphloroglucinols from Dryopteris 824

Table 1[ APCI!CAD� mass spectral data for phloroglucides 0Ð2 ðm:z "rel[ int[# $Ł

Compound Mr XH¦ Y?H¦ X?H¦ YH¦

Para!aspidin0!AA 393 086 "099# 198 "87# 198 "87# 086 "099#

Desaspidins1!AA 289 086 "01# 084 "099# 198 "47# 072 "82#1!AP 393 086 "099# 198 "77# 198 "77# 086 "099#1!AB 307 086 "05# 112 "75# 198 "21# 100 "099#1!PB 321 100 "099# 112 "77# 112 "77# 100 "099#

Albaspidins2!AA 393 086 "099# 198 "57# 198 "57# 086 "099#2!AP 307 086 "099# 112 "01# 198 "8# 100 "80#2!AB 321 086 "099# 126 "12# 198 "14# 114 "86#2!PP 321 100 "099# 112 "25# 112 "25# 100 "099#2!PB 335 100 "88# 126 "124# 112 "14# 114 "099#2!BB 359 114 "099# 126 "45# 126 "45# 114 "099#

� Atmospheric pressure chemical ionization!collision activated decomposition[ Compare Fig[ 1 for structures of fragmentions[ Minor fragments are listed in the Experimental section[

$ Principal ions are printed in bold type face[ See also text for further explanation[

Fig[ 1[ APCI!CAD mass fragmentation of desaspidins "top# and albaspidins "bottom#[ For intensities of X and Y cleavageions\ see Table 1[

therein#[ By FAB mass spectrometry\ Wide�n et al[ ð06Łwere able to obtain ðMŁ¦s of albaspidins up to thehexamer[ Dimeric albaspidins yielded similar spectra

with APCI!MS in our study[ Without further exam!ples of APCI!MS analysis of olgomeric phloro!glucinols it is di.cult to estimate the upper limit of

ECKHARD WOLLENWEBER et al[825

the molecular range up to which molecular infor!mation can be obtained by APCI!MS[ Clearly\ APCIhas the advantage over FAB mass spectrometry inthat the lower mass region is not obscured by matrixions and that it is well!suited for coupling with ana!lytical HPLC[ Although vaporization of the HPLCe/uent involves heat\ artifacts due to Rottleronerearrangement\ often seen in EI mass spectra of asym!metric oligomeric acylphloroglucinols ð07Ł\ were notobserved in our APCI mass spectra of asymmetricalbaspidins[ For instance\ APCI of 2!PB ðMH¦ at m:z336Ł did not give rise to expectable Rottlerone artifactswith m:z 322 and m:z 350[ Hypothetically\ Rottleronerearrangement of an albaspidin X0CH10Y may giverise to two symmetric albaspidins X0CH10X andY0CH10Y by recombination of a neutral fragmentX with X?H¦ or Y with Y?H¦ in the ion source "cfFig[ 1#[

Localization and distribution of acylphloro`lucinols inDryopteris

Light microscopic examinations of the three fernspecies revealed the presence of glandular trichomeson their leaf surfaces[ The ultrastructure of the leafglands of D[ villarii and C[ robertiana was investigatedby scanning electron microscopy of fresh material"Fig[ 2#[ The glandular trichomes of both species weresimilar[ They appear to be unicellular trichomes withround to orbicular heads "Fig[ 2"c##\ thus cor!responding with the glandular trichomes depictedearlier for D[ sparsa ð08Ł or reported for Arachnoidesspecies ð19Ł[ According to these authors\ the exudateis deposited on the distal end of these trichomes\ pos!sibly between cell wall and cuticle ð10Ł[ In view of theearlier discussed correlation between ~avonoid agly!cones and the presence of secretory structures ð11Ł\ theleaf surface phloroglucinols of both Dryopteris fernsare presumably produced by their glandular trich!omes[ This hypothesis also parallels earlier obser!vations that Dryopteris phloroglucinols are excretedalong with other resinous materials by internal glandspresent in the rhizomes and petiolar bases ð12Ł[ Draw!ings as well as electron microscopic pictures showingthe internal glandular hairs of D[ assimilis Walker" �D[ expansa "Presl# Fraser!Jenkins and Jermy# werepublished in 0868 ð10Ł[ In that species\ glandular hairsof 59Ð89 mm length were observed in large inter!cellulars in the rhizomes[ It was demonstrated that thephloroglucinols were biosynthesized in the glandularcytoplast and subsequently accumulated in a sub!cuticular excretion layer[ Some years earlier\ a draw!ing of the external secreting hairs of D[ fra`rans "L[#Schott was published ð13Ł[ These glandular hairs occuron the epidermis of the rhizomes and petiolar bases[They were said to contain much secreted materialbetween the cuticle and the outer layer of the cell[Internal secreting hairs were not round in this species[Similar drawings exist for D[ campyloptera "Kuntze#

Clarkson\ D[ expansa\ and D[ intermedia "Muehl# A[Gray ð14Ł[

Comparison of the leaf surface phloroglucinolsidenti_ed in this study with rhizome phloroglucinolsreported for D[ villarii and D[ ar`uta ð15Ł indicatesthat the leaves di}er from the rhizomes with regard tophloroglucinol composition[ Wide�n et al[ ð15Ł detected~avaspidic acids "BB and AB#\ para!aspidin BB\ and_lixic acids "BBB and ABB# in the rhizomes of bothD[ villarii and D[ ar`uta[ In D[ villarii rhizomes\ theseacylphloroglucinols were found along with smallamounts of desaspidin BB and albaspidin BB[ Thequalitative compositions of D[ villarii and D[ ar`utarhizomes from di}erent sources proved to be similarð15Ł[ At this point it is unknown to what extent thequalitative di}erences are associated with organ toorgan variability[ Comparative analyses of leaves andrhizomes from individual plants will shed more lighton this question[ On the other hand\ the absence ofacylphloroglucinols on the leaf surface of C[ robertianais in agreement with an earlier report of absence ofacylphloroglucinols in the rhizomes of this fern ð8Ł[

At this point we can only speculate about the eco!physiological function of leaf surface phloroglucinolsof Dryopteris[ It is conceivable that the distribution ofleaf surface acylphloroglucinols reaches beyond thesetwo species of Dryopteris[ As with externally accumu!lated ~avonoid aglycones\ the aspect of localizationof phloroglucinols in the plant certainly deservesattention when biological e}ects are of interest[ Forexample\ literature reports on the neurotoxic e}ectsof acylphloroglucinols in cattle "drowsiness and blind!ness# eating the rhizomes and leaves of male fern "D[_lix!mas# are exclusively focused on the rhizomes ð16Ł[

EXPERIMENTAL

Plant material

Dryopteris villarii was collected by R[ Mues "Saar!bru�cken# in August 0884 and by E[ Wollenweber"Darmstadt# in October 0885 at the Schynige Platte inthe Berner Oberland\ Switzerland "alt[ ca 0859 m#[Dryopteris ar`uta was collected by J[ N[ Roitman on03 March\ 0883\ in oak woodland near the El CariseOaks Campground on hwy 63\ 12 miles east of Inter!state 4 "San Juan Capistrano#"elev[ ca 799 m#\ Riv!erside Co[\ CA[ Currania robertiana was collected byR[ Mues in July 0884 near Ilanz in Graubu�nden\ Swit!zerland and by E[ Wollenweber in the BotanischerGarten der TH Darmstadt[ Fronds were carefullyclipped in the _eld and air!dried in paper bags[ Vou!chers are kept at the Universities of Saarbru�cken andDarmstadt[

NMR

0H "599 MHz# and 02C "049[8 MHz#^ CDCl2 at roomtemp[ Albaspidins 1!PB and 1!BB were run at 172 K

Leaf surface acylphloroglucinols from Dryopteris 826

Fig[ 2[ SEM views of glandular trichomes on the leaf surface of "a# D[ villarii "epidermal walls shrank on drying#^ "b# C[robertiana "lower leaf surface\ survey#^ "c# C[ robertiana "detail#[ For magni_cation see scale bar on the photographs[

to reduce keto!enol tautomerism[ TMS and CHCl2resonances "dH 6[15 ppm\ dC 62[12 ppm# were usedas int[ chemical shift references[ 0HÐ02C HMQC and

HMBC experiments were performed using standardpulse sequences[ Spectral widths of 19 and 119 ppmwere used in the H and C dimensions\ respectively[

ECKHARD WOLLENWEBER et al[827

Mass spectrometry

APCI!MS] PE Sciex API III plus triple quadrupoleinstrument[ Samples were introduced by loop injec!tion or by HPLC via a heated nebulizer interface keptat 399> which heats the column e/uent to ca 019>[Ionization of the analyte vapour mixt[ was initiated bya corona discharge needle at ca 5 kV and a dischargecurrent of ca 2 mA[ The ori_ce plate voltage was set at44Ð54 V in the positive ion mode[ MS!MS experimentswere performed with ArÐN1 "8]0# as collision gas at athickness of ca 0[8×0903 atoms−1[ The collisionenergy was 04 V[ Other operating conditions werestandard[

Isolation and chromato`raphic procedures

Field!collected air!dried materials as well as freshleaves from the Botanical Garden were brie~y rinsedwith Me1CO to dissolve the exudates[ After evapn ofthe solvent\ the residues of D[ villarii and D[ ar`utawere redissolved in a small amount of boiling MeOH\cooled to −07> and centrifuged to eliminate the majorpart of the lipophilic constituents[ The supernatantwas chromatographed on polyamide SC!5\ eluted withtoluene and increasing amounts of MeCOEt andMeOH[ In C[ robertiana the residue was directly sub!jected to CC on acetylated polyamide[ Fractions weremonitored by TLC on polyamide "DC!00\ Macherey!Nagel# with solvents A ðpetrol "bp099Ð039>#ÐtolueneÐMeCOEtÐMeOH 01]5]0]0Ł\ B ðtolueneÐpetrol"bp099Ð039>#Ðmethyl ethyl ketoneÐMeOH 01]5]1]0Łand C "tolueneÐMeCOEtÐMeOH 01]4]2# and on sil!ica with solvents D "tolueneÐMeCOEt 8]0# and E"tolueneÐdioxaneÐHOAc 07]4]0#[ Chromatogramswere viewed under UV "255 nm# before and afterspraying with Natursto}reagenz A[ Identi_cation of~avonoids was achieved by co!TLC with authenticmarkers available in E[W|s lab[

HPLC

Analyt[ RP!HPLC] 4 mm LiChrospher RP!07column "149×3 mm# at 0 ml min−0 with a linearsolvent gradient from 79) to 84) MeCN in 0) aq[HCOOH over 04 min followed by a further 04 min at84) MeCN[ Semi!prep RP!HPLC] 09mm EconosilRP!07 column "149×09 mm# at 4 ml min −0[ D[ villariiphloroglucinols were separated isocratically with 89)MeCN in 0) aq[ HCOOH[ For semi!prep isolationof D[ ar`uta phloroglucinols\ a linear solvent gradientfrom 79) to 84) MeCN in 0) aq[ HCOOH over04 min was used\ followed by a further 04 min at 84)MeCN[ The UV trace was recorded at 179 nm[ Peakfrs were collected manually and evapd on a rotavaporand by lyophilization[ Milligram or sub!mg amountswere obtained as white to cream!coloured amorphouspowders[

Para!aspidin!AA "0!AA#[ 0H NMR] d 0[44 "5H\s\ × C"Me#1#\ 1[03 "2H\ s\ arom[ Me#\ 1[60 and 1[63

"each 2H\ s\ 1×acetyl Me#\ 2[46 "1H\ br\ s\ H!6#\2[63 "2H\ s\ OMe#\ 09[99\ 00[49\ 04[47 and 07[30 "s\3×OH#^ APCI!MS\ m:z "rel[ int[#] 394 ðMHŁ¦"099#[APCI!CAD!MS] Table 1 and m:z 276ðMH−H1OŁ¦"09#\ 068 ðYHÐH1OŁ¦ "11#[

Desaspidin "1!AA#[ 0H NMR] d 0[43 "5H\s\ × C"Me#1#\ 1[52 and 1[62 "each 2H\ s\ 1×acetylMe#\ 2[43 "1H\ br s\ H!6#\ 2[74 "2H\ s\ OMe#\ 5[94"0H\ s\ H!4?#\ 8[81\ 00[24\ 05[12 and 07[30 "s\ 3×OH#^APCI!MS\ m:z "rel[ int[#] 280 ðMHŁ¦"099#[ APCI!CAD!MS] Table 1 and m:z 262 "MHÐH1OŁ "5#\ 054ðYHÐH1OŁ¦ "11#[

Desaspidin AP "1AP#[ 0H NMR] d 0[06 "2H\ t\J � 6[1 Hz\ H!09?#\ 0[45 "5H\ s\ ×C"Me#1#\ 1[62 "2H\s\ acetyl Me#\ 2[94 "1H\ m\ H!8 ?#\ 2[42 "1H\ br s\H!6#\ 2[74 "2H\ s\ OMe#\ 5[95 "0H\ s\ H!4?#\ 8[86\00[20\ 05[23 and 07[30 "s\ 3×OH#[ APCI!MS\ m:z"rel[ int[#] 394 ðMHŁ¦ "099#^ APCI!CAD!MS] Table 1and m:z 276 ðMHÐH1OŁ¦ "3#\ 068 ðYHÐH1OŁ¦ "10#[

Deaspidin AB "1!AB#[ 0H and 02C NMR] Table 0^APCI!MS\ m:z "rel[ int[#] 308 ðMHŁ¦ "099#^ APCI!CAD!MS] Table 1 and m:z 390 ðMHÐH1OŁ "4#\ 082ðYHÐH1OŁ¦ "13#[

Desaspidin PB "1!PB#[ 0H NMR] d 9[88 "2H\ t\J � 6[3 Hz\ H!00?#\ 0[06 "2H\ t\ J � 6[1 Hz\ H!09#\0[43 "5H\ s\ ×C"Me#1#\ 0[69 "1H\ m\ H!09?#\ 1[82Ð2[96"3H\ m\ H!8 and H!8?#\ 2[42 "1H\ br s\ H!6#\ 2[74 "2H\s\ OMe#\ 5[94 "0H\ s\ H!4?#\ 8[83\ 00[22\ 05[27 and07[36 "s\ 3×OH#^ APCI!MS\ m:z "rel[ int[#] 322ðMHŁ¦ "099#[ APCI!CAD!MS] Table 1 and m:z 304ðMHÐH1OŁ¦ "3#\ 252 ðMH−O1C1CH0C1H4Ł¦ "1#\082 ðYHÐH1OŁ¦ "07#[

Albaspidin AA "2!AA#[ APCI!MS\ m:z "rel[ int[#^394 ðMHŁ¦ "099#^ APCI!CAD!MS] Table 1 and m:z252 ðMH−O1C1CH1Ł¦ "5#\ 068 ðXHÐH1OŁ¦ "32#\044 ð_licinic acid¦HŁ¦ "16#[ This product was notisolated but was kindly provided by Prof[ C[ J[ Wide�nfor comparison purposes[

Albaspidin AP "2!AP#[ APCI!MS\ m:z "rel[ int[#] 308ðMHŁ¦ "099#[ APCI!CAD!MS] Table 1 and m:z 266ðMHÐO1C1CH1Ł¦ "1#\ 252 ðMHÐO1C1CH0CH2Ł¦ "5#\ 068 ðXHÐH1OŁ¦ "3#\ 082ðYHÐH1OŁ¦ "00#\ 044 ð_licinic acid¦HŁ¦ "3#[

Albaspidin AB "2!AB#[ APCI!MS\ m:z\ "rel[ int[#]322 ðMHŁ¦ "099#[ APCI!CAD!MS] Table 1 and m:z280 ðMHÐO1C1CH1Ł¦ "3#\ 252 ðMHÐO1C1CH1C1H4Ł¦ "11#\ 196 ðYHÐH1OŁ¦ "34#\ 068ðXHÐH1OŁ¦ "03#\ 044 ð_licinic acid ¦ HŁ¦ "03#[

Albaspidin PP "2!PP#[ APCI!MS\ mz "rel[ int[#] 322ðMHŁ¦ "099#[ APCI!CAD!MS] Table 1 and m:z 266ðMHÐO1C1CH0CH2Ł¦ "05#\ 082 ðXHÐH1OŁ¦ "31#\044 ð_licinic acid ¦ HŁ¦ "03#[

Albaspidin PB "2!PB#[ 0H and 02C NMR] Table 0^APCI!MS\ m:z "rel[ int#] 336 ðMH#¦ "099#[ APCI!CAD!MS] Table 1 and m:z 280 ðMHÐO1C1CH0CH2Ł¦ "05#\ 266 ðMHÐO1C1CH0C1H4Ł¦ "17#\ 196 ðYHÐH1OŁ¦ "15#\ 082ðXHÐH1OŁ¦ "15# ð_licinic acid ¦ HŁ¦ "03#[

Albaspidin PB "2!PB#[ 0H and 02C NMR] Table 0^APCI!MS\ m:z "rel[ int#] 350 ðMHŁ¦ "099#[ APCI!

Leaf surface acylphloroglucinols from Dryopteris 828

CAD!MS] Table 1 and m:z 280 ðMHÐO1C1CH0C1H4Ł¦ "52#\ 196 ðXHÐH1OŁ¦ "44#\ 044ð_licinic acid ¦ HŁ¦ "08#[ 2!BB was identical withauthentic albaspidin BB by HPLC and APCI!MS!MS[

Aspidinol B "3#[ "1?\5?!Dihydroxy!3?!methoxy!2?!methyl!l?!butyrophenone#[ 0H NMR] d 0[99 "2H\ t\J � 6[1 Hz\ H!3#\ 0[62 "1H\ m\ H!2#\ 1[91 "2H\ s\ Me!2?#\ 2[97 "1H\ t\ J � 6[1 Hz\ H!1#\ 2[70 "2H\ s\ OMe!3?#\ 4[85 "0H\ s\ H!4?#\ 8[58 and 09[13 "1? and 5?!OH#^02C NMR] d 6[1 "Me!2?#\ 03[0 "C!3#\ 07[4 "C!2#\ 35[2"C!1#\ 44[8 "OMe!3?#\ 093[2 "C!2?#\ 81[0 "C!4?#\ 049[4"C!0?#\ 059[2 "C!1?#\ 050[3 "C!5?# 052[5 "C!3?#\ 193[5"C!0#[ APCI!MS\ m:z "rel[ int[#\ 114 ðMHŁ¦ "099#^APCI!CAD!MS\ m:z] 114 ðMHŁ¦ "099#\ 196 ðMHÐH1OŁ¦ "68#\ 072 "34#\ 068 ð196−COŁ¦ "69#\ 040 ð196−1COŁ¦ "69#\ 002 "25#[ Compound 3 was identical withauthentic aspidinol B by HPLC and APCI!MS!MS[

Acknowled`ements*E[W[ wishes to thank Prof[ Dr RMues "Saarbru�cken# who drew his interest to thesefern species and supplied some of the material studiedhere[ Thanks are also due to Prof[ Dr J[ Schneller"Zu�rich# who supplied two plants of Currania rober!tiana\ to Dr J[ N[ Roitman "Albany\ CA# who col!lected Dryopteris ar`uta\ to Mrs Marion Do�rr "Darm!stadt# for technical assistance^ to Mrs H[ Wilhelmiand Prof[ Dr W[ Barthlott "both at the BotanischesInstitut der Universita�t Bonn# for SEM photographsand permission to publish them\ and to Prof[ Dr V[L[ Hsu "OSU\ Corvallis# for access to NMR facilities[We further wish to thank Prof[ Dr C[!J[ Wide�n"Helsinki\ Finland# and Prof[ Dr P[ AÝyra�s "Turku\Finland# for a gift of marker compounds\ for reprintsand for valuable discussions[

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