Biochemical Systematics and Ecology 28 (2000) 229}253 Distribution and taxonomic implications of some phenolics in the family Lamiaceae determined by ESR spectroscopyq Jens A. Pedersen* University of British Columbia, Department of Botany, Vancouver B.C., Canada V6T 2B1 Received 16 December 1998; accepted 15 February 1999 Abstract The dihydric phenolics from the leaves of 365 specimens representing 355 species and varieties of 110 genera of Lamiaceae (Labiatae) have been examined as semiquinones by ESR spectroscopy. Of these 89 genera comprising 194 species belong to the Lamiaceae, whereas 21 genera comprising 161 species have been transferred from the Verbenaceae in the most recent classi"cation. Two chemical characters give strong support to the subfamily division of the Lamiaceae as recognised by Erdtman (1945). (Svensk Bot. Tidskr. 29, 279}285) and others. Rosmarinic acid was observed in 110 species out of 127 studied and con"ned to the subfamily Nepetoideae sensu Erdtman. These species produce tricolpate pollen grains. 3,4- Dihydroxyphenylethanoid glycosides were observed in 111 species all con"ned to Lamioideae sensu Erdtman with hexacolpate pollen grains. The acid and the phenylethanoid glycosides were found to be mutually exclusive, apart from one species, Teucrium scorodonia. A compound tentatively assigned as a b-hydroxy-(3,4-dihydroxyphenyl) ethanoid glycoside, was observed in 49 species, solely con"ned to subfamily Lamioideae. Furthermore, rosmarinic acid was observed in Aegiphila mollis and in Hymenopyramis brachiata, both belonging to Lamioideae. No phenylethanoid glycosides were observed from any of 5 Hymenopyramis or from any of 30 species of Vitex. The latter result questions the transfer of the genus to Lamiaceae, but strengthens its isolated position. Chlorogenic acid was observed in 34 species and protochatechuic acid in 16 species. Hydroquinone is scattered in the family (4 species) and thymohydroquinone was observed in 7 species all belong to the Nepetoideae. ( 2000 Elsevier Science Ltd. All rights reserved. q This paper was presented at the North American Phytochemical Society Meeting, 1998, Pullman, Washington State University. * Corresponding author. Dr. Jens A. Pedersen, Aarhus University, Department of Chemistry, Langelandsgade 140, DK-8000 Aarhus C, Denmark. E-mail address: jap@kemi.aau.dk (J.A. Pedersen) 0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 5 - 1 9 7 8 ( 9 9 ) 0 0 0 5 8 - 7 230 J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 Keywords: Lamiaceae; Verbenaceae; Rosmarinic and Chlorogenic acid; Dihydroxy-phenylethanoid glycosides; Phenolics; ESR; Pollen morphology; Chemotaxonomy 1. Introduction The family Lamiaceae has a cosmopolitan distribution and highly varied morphological characters. In 1789 de Jussieu named the family Lamiaceae. A major subfamilial classi"cation was made by Bentham (1876) with revisions introduced by Briquet (1895}97). Briquet's classi"cation has been the most widely used system for the family. A number of studies, however, has clearly indicated the need for a reevaluation of both subfamilial and tribal division of the family as well as its limits. Recent evaluations of Lamiales suggest removal of a number of genera from Verbenaceae to Lamiaceae, leading to much increased limits for the latter. Such a transfer was already suggested by Junell (1934). In the most recent classi"cation the family comprises about 252 genera and 6700 species. Leitner (1942) revealed a correlation between the number of nuclei in Lamiaceae pollen grains and number of colpi in their exines, viz. tricolpate grains are binucleate and hexacolpate are trinucleate. These palynological features led Erdtman (1945) to subdivide the family into two subfamilies Lamioideae with tricolpate 2-nucleate pollen grains and Nepetoideae with hexacolpate 3-nucleate pollen grains. Erdtman's subdivision has since gained increasing support in a number of papers, e.g. by Wunderlich's comprehensive pollen survey (Wunderlich, 1967) or Cantino and Sanders analysis based on 11 morphological and chemical characters (Cantino and Sanders, 1986). Harborne (1966) indicated that rosmarinic acid appears to be restricted to species belonging to Nepetoideae sensu Erdtman. Since then other chemical characters have been studied to support subdivisions made for the family (TomaH s-BarbaraH n and Gil, 1992; Cole, 1992). The present study was undertaken in order to see whether the actual distribution of simple phenolics would give evidence of taxonomic signi"cance for this large and varied family. A further impetus was given by the fact, that many phenylethanoid glycosides, widely distributed in families of Lamiales, have been found to have various biological activities, see e.g. JimeH nez and Riguera (1994). Maps of their distribution and general occurrence are accordingly of broad interest. To handle the rather large number of specimens we had available (365 specimens comprising 110 genera), we performed a screening by means of Electron Spin Resonance (ESR) spectroscopy. The technique is ideal to detect and distinguish compounds such as rosmarinic acid, chlorogenic acid and 3,4-dihydroxyphenyl ethanoid glycosides, compounds frequently reported from Lamiaceae species. By the technique quinones are reduced and aromatic compounds with free o-or p-dihydroxy groupings oxidised, in base, to semiquinone radicals and detected and identi"ed, absolutely or partly in the crude extract by way of their unique spectra (Pedersen, 1978). The technique is advantageous being rapid and requiring tiny amounts of plant material, e.g. detached leaves of "ngernail size (Pedersen and "lgaard, 1982). It should be emphasised, however, that J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 231 quinols not having o-or p-dihydroxy groups are not detected, e.g. if one of the hydroxy groups is replaced by an alkoxy group this particular compound is ignored by the spectrometer. We report here the results obtained from our ESR study. Since we had available, in addition to our Lamiaceae specimens, a large number of Verbenaceae specimens we were interested to see whether our "ndings for 21 genera of this family supported their transfer from Verbenaceae to Lamiaceae, as suggested recently (Cantino et al., 1992). Accordingly, we have included our results of pertinent Verbenaceae. 2. Results and discussion 2.1. ESR results 2.1.1. Reaction I Two prominent semiquinone spectra are frequently observed from specimens of Lamiaceae by aerial oxidation in dilute alkali (see Material and Experimental). One is derived from rosmarinic acid (Fig. 1A) clearly identi"ed by the fact that an identical spectrum is observed from an authentic sample of this compound (Pedersen, 1978). The other is derived from radicals of 3,4-dihydroxyphenylethanoid glycosides (normally observed as one spectrum, Fig. 2A), since an identical spectrum in this case is obtained from an authentic sample of acteoside. We shall henceforth abbreviate 3,4-dihydroxyphenylethanoid glycosides as dHPhGs. Spectra similar to 24 line spectrum of Fig. 2A are often observed by the ESR technique applied to crude plant extracts, in particular from species belonging to families of Lamiales. (Only 18 lines are seen due to accidental overlap). Thus, 24 line spectra were observed in 217 specimens of the Gesneriaceae family in a comprehensive study of this family (Kvist and Pedersen, 1986). In these the spectra are derived from dHPhGs such as acteoside and conandroside. Observation of the 24 line spectrum is direct proof that the compound present in the extract belongs to the group of dHPhGs. The identity of the glycoside moiety is not obtainable from the ESR spectrum. However, the presence of two di!erent dHPhGs in an extract of Conandron ramondioides was substantiated by advanced simulations of the observed ESR spectrum (Kvist and Pedersen, 1986). Both acteoside and conandroside are known to be present in this particular plant (Nonaka and Nishioka, 1977). Acteoside and a number of related compounds have previously been isolated from specimens of Lamiaceae (see e.g. JimeH nez and Riguera, 1994). Table 1 gives the structures of some of these compounds, which all possess a 3,4-dihydroxyphenylethyl moiety. Any of these compounds are amenable to yield spectra similar to the one of Fig. 2A, when transferred to the semiquinone state. Acteoside ("verbascoside), however, is usually the most abundant among the dHPhGs found in a particular species and its ESR spectrum will accordingly mask spectra from less abundant ones in the extract. We shall collectively designate dHPhGs with an A, we also use A to represent the semiquinone radicals of the corresponding compounds in the extract. 232 J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 Fig. 1. The ESR semiquinone spectrum of (a) rosmarinic acid from Peltodon radicans of (b) hydroquinone from Salvia hispanica and (c) thymohydroquinone from Monarda clinopodia all obtained from crude alcoholic leaf extracts. J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 233 Fig. 2. The ESR semiquinone spectrum of (a) from radicals of 3, 4-dihydroxy-phenylethanoid glycosides and of (b) from a radical tentatively assigned as derived from a b-hydroxy -(3, 4-dihydroxyphenyl) ethanoid glycoside (R might be an alkoxy group and R@ at least of the size of a monosaccharide). Both obtained from crude alcoholic leaf extracts of Hemiandra pugens. 234 J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 Table 1 Structures of some 3,4-dihydroxyphenylethanoid glucosides 3, 4-Dihydroxyphenylethanoid glycoside R1 R2 R3 R4 References Deca!eoylacteoside Acteoside H H H Ca!eoyl H H H H Isoacteoside Ca!eoyl H H H Leucosceptoside A H Feruloyl H H Leonoside A Lavandulifolioside (Stachyoside A) Phlinoside A Phlinoside B Phlinoside C Premcoryoside Teucrioside H H Feruloyl Ca!eoyl Ara Ara H H H H H IRI(X:~) H Ca!eoyl Ca!eoyl Ca!eoyl Ca!eoyl Ca!eoyl Glu Xyl Rha H Lyx H H H H H Nishimura et al. (1991) Calis et al. (1990; 1992a, b), Nishimura et al. (1991) Nishimura et al. (1991), Miyase et al. (1982) Calis et al. (1992a), Nishimura et al. (1991), Miyase et al. (1982) Calis et al. (1992b) Calis et al. (1992a, b), Nishimura et al. (1991) Calis et al. (1990) Calis et al. (1990) Calis et al. (1990) Otsuka et al. (1993) Gross et al. (1988) H Ca!eoyl H OH Nishibe et al. (1982) b-Hydroxyacteoside (Campneoside II) It should be noticed that naturally occurring esters of dihydroca!eic acid give rise to 24 line spectra which seem to be identical to the spectrum of A. However, recourse to their coupling constants indicates clear di!erences and the compounds are easily distinguished (Pedersen and "lgaard, 1982; Kvist and Pedersen, 1986). We shall designate rosmarinic acid with an R and, as before use R as representing the semiquinone radical of the acid. It should be noticed that the ca!eoyl moiety, present in R and in some of the A compounds, does not possess the unpaired electron in Reaction I, viz. the semiquinone radical of the ca!eoyl group is not observed for these compounds. On the other hand compounds containing a ca!eoyl moiety as the only group with a 3,4-dihydroxyphenyl structure are easily observed by the ESR technique. Thus, chlorogenic acid yields a spectrum easily distinguished from either A or R (see, e.g. Pedersen, 1978). We have observed chlorogenic acid in a number of specimens and shall designate it as C. It should be noticed that neochlorogenic acid in the ESR routine procedure gives rise to an identical spectrum. J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 235 Table 2 ESR data of semiquinone radicals observed from species of Lamiaceae Radical Structure ESR hyper"ne splitting constants (Gauss) A A1 C K R R1 H T P B1 Acteoside 6-Hydroxyacteoside Chlorogenic acid Ca!eic acid Rosmarinic acid 6-Hydroxyrosmarinic acid! Hydroquinone Thymohydroquinone Protocatechuic acid b-hydroxy-(3,4-dihydroxyphenyl)ethanoid glycoside" 6-hydroxyca!eic acid Unknown Unknown 0.45 0.58 0.65 0.28 0.52 0.21 2.36 1.95 0.80 0.54 R9 X5 X6 (H), 1.00 (H), 0.96 (H), 1.17 (H), 1.23 (H), 1.06 (H), 0.54 (4 H) (H), 1.79 (H), 1.35 (H), 1.43 (H), (H), (H), (H), (H), (H), 3.60 3.08 1.17 1.27 2.21 1.02 (H), 3.10 (2H) (2H) (H), 2.32(H), 2.60 (H) (H), 2.36 (H), 2.83 (H) (H), 3.30 (H), 3.80 (H) (H), 2.40 (H), 4.10 (H) (H), 1.43 (H), 2.16 (3H) (H), 3.30 (H) (H), 3.75 (H), 0.21 (2H) 0.39 (H), 1.35 (H), 1.78 (H), 2.90 (H) 0.20 (H) 0.61 (H), 0.99 (H), 5.79 (H) ! The analysis of the complex spectrum of R1 is incomplete, viz. a"2.40 and 4.10 might read 2.95 and 3.55. " Tentatively assigned structure (cf. Fig. 2B) Other compounds of scattered occurrence have been observed in reaction I. Thus, hydroquinone, designated H, has been observed in four specimens. The spectrum of the semiquinone gives rise to a characteristic 5-line pattern (1:4:6:4:1, Fig. 1B). Strong signals of thymohydroquinone, designated T, has been observed in 7 specimens, (Fig. 1C). Finally, protochatechuic acid, designated P and furnishing an 8-line spectrum, has been observed in 11 species of Vitex and in "ve specimens scattered in the family. H, P and T are all absolutely identi"ed from their ESR spectra. The hyper"ne splitting constants obtained for the observed semiquinone radical spectra are collected in Table 2. 2.1.2. Reaction II If we perform the oxidation on the ethanolic extract in stronger bases (see Material and Experimental) we might observe degradation/hydroxylation products of compounds present in the extract. Thus, rosmarinic acid eventually leads to a product selectively hydroxylated at the C-6 phenyl carbon of the dihydroca!eic acid unit. A glance at Table 3 shows that this artefact, designated R , is observed only in extracts 1 containing R. The absence of R in some extracts clearly containing R is due to the 1 fact that the hydroxylation process and subsequent transfer to the radical state (semiquinone) are delicate reactions the optimum conditions of which may vary from extract to extract. Furthermore, a number of the Lamiaceae specimens were studied some time ago, with no particular e!ort made to observe R . Similarly, the alkaline 1 (stronger base) oxidation of the A-compound leads to the selective hydroxylation at the C-6 phenyl carbon of the 3,4-dihydroxyphenylethyl mooiety. This artefact, designated A , may eventually be observed as the corresponding semiquinone radical, 1 exhibiting a characteristic 12 line spectrum. As before A is observed only in those 1 236 J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 Table 3 Occurrence of Dihydroxyphenolics in studied taxa of Lamiaceae R A I. Ajugoideae Ajuga chia Schreb. A. remota Benth. A. reptans L. II. Chloanthoideae tr Dicrastylis gilesii v. bogotensis F. Muell. tr D. verticillata J.M. Black Hemiandra pugens R. Br. Hemigenia pritzelli S. Moore tr Lachnostachys coolgardiensis S. Moore tr Newcastelia cephalantha v. cephalantha F. Muell. Prostanthera ovalifolia R. Br. tr Tectona grandis L.f. T. grandis L.f. Westringia rosmariniformis Sm. III. Lamioideae Achyrospermum densiyorum Blume A. laterale Baker Ballota acetabulosa (L.) Benth. B. acetabulosa (L.) Benth. B. nigra L. B. pseudodicthamnus Benth. Betonica ozcinalis L. Colquhounia coccinea Vall. &tomentosa' Galeopsis speciosa Mill. Lamium album L. L. amplexicaule L. L. purpureum L. Leonotis dysophylla Benth. L. leonitis (R. Br.) Ait. Leonurus cardiaca L. L. marrubiastrum L. Leucas biyora (Vahl) Benth. L. mollissima Wall. v. chinensis Benth. Leucophae dasygnaphala Webb. Marrubium astracanicum Jacq. M. peregrinum Melittis melissophyllum L. Molucella laevis L. Panzeria argyracea Kuprion Paraphlomis grasilis Kudo Phlomis bovie de NoeH P. fruticosa L. P. italica L. P. russeliana Benth. C B 1 A1 R1 R9 X6 X5 other Tri Hexa Voucher # # # UCo Kew Den # # # # # # AaUH AaUH UBCH UBCH AaUH # # # # # # AaUH Kew AaUH BAa UCoH # # # # # # # # # # # # # # # # # # # # # # # # # # # # # AaUH Kew UCo UBC BAa UCo BAa UCo Den Den Den Den Kew Kew BAa BAa AaUH UBCH AaUH AaUH UBC UCo UCo AaUH AaUH UCo UCo UCo UCo # # # # ## t ## # ## ## # # # ## ### ## # # # # # # # # ## # # # # # # ## # # # t # # # # p # # # t # # # # # # # # J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 237 Table 3*continued R P. viscosa Poir. Physostegia denticulata (Ait.) Britton P. viginiana (L.) Benth. Pseuderemostachys severtzovii (Herd.) Popov Sideritis dasygnaphala Clos S. macrostachya Poir. S. montana L. Stachys albomentosa T.P. Ramamoorthy S. palustris L. S. silvatica L. Stenogyne calaminthoides A. Gray S. purpurea H. Mann IV. Nepetoideae IV. 1. Mentheae Acinos arvensis (Lam.) Dandy Agastache cana (Hook.) W. & S. A. foeniculum Kuntze Calamintha nepeta Savi Cedronella canariensis W. & B. Cleonia lusitanica L. Clinopodium vulgare L. Conradina verticiliata Jennison Cuminia fernandeziana v. fernandezia (Colla) Harley Cunila ssp. Dracocephalum moldavica L. D. moldavica L. &album' D. parviyorum Nutt. D. ruyschiana L. D. thymiyorum L. Glechoma hederacea L. Hyssopus ozcinalis L. &album' Lallemantia canescens Fisch. & May. Lepechinia yoribunda (Benth.) Epl. L. schiediana (Schlechtd.) Vatke Lycopus europaeus L. L. exaltatus L.f. Meehania cordata (Nutt.) Britt. Melissa ozcinalis L. Mentha aquatica L. M. arvensis L. M. galtefossei Maire M. rotundifolia (L.) Huds. M. satureiodies R. Br. M. spicata L. Meriandra bengabensis Benth. A C B 1 A1 R1 R9 X6 X5 other Tri Hexa Voucher # # # # # # t # # ## # # # # t H # # # # # ## # # ## # ## # ## # # # t # # # ## # # ## # # # # # ## # t # # # # # ## # # ## # ## ## # # # # # # # # H # # # BAa UCo BAa # # # # # AaUH UCo UCo BAa UBC # # # # Den Den AaUH AaUH # # # # # # # # AaUH UCo UCo UCo UCo UCo BAa UBC # # # # # # # # # # # # # # # # # # # # # # # Kew Kew UCo UCo UBCH BAa UCo Den BAa UCo UCoH Kew Den BAa AaUH Den Den Den UCoH BAa Kew Den AaUH * continued 238 J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 Table 3*continued R Micromeria benthamii W. & B. M. dalmatica Benth. M. cf. thymifolia Minthostachys mollis (Kunth) Griseb. Monarda clinopodia L. M. didyma L. M. xstulosa L. Monardella lanceolata A. Gray M. linoides A. Gray M. odoratissima Benth. Nepeta cataria L. N. kokanica Regel N. macrantha Ledeb. ex Hook. N. mussini Spreng. N. nepetella L. N. parnassica Heldr. & Sart. N. sibirica L. Origanum onites L. O. prismaticum O. sipyleum L. O. tyttanthum Gontsch. O. vireus Gren. & Godr. O. vulgaris L. Peorvskia atriplicifolia Benth. Prunella grandiyora (L.) Scholl P. hyssopifolia L. P. vulgaris L. P. vulgaris L. P. vulgaris L. P. webbiana Hort. ex N. Taylor Rhabdocaulon lavanduloides Epl. Rosmarinus ozcinalis L. Salvia algeriensis Desf. S. aurea L. S. dichroantha Stapf S. forskohlei L. S. gesneriyora Lindl. & Paxton S. hispanica L. S. lyrata L. S. nilotica Murr. S. pratensis L. S. ringens Sibth. & Sm. S. tilefolia Vahl Satureja hortensis L. S. montana L. S. thymbra L. S. vulgaris (L.) Fritsch. Thymus nitens Lamotte T. polygoides Zataria multiyora Boiss. # # # # # # # # # # # # # # # ## # # # # t # # ## ## ## # # # t # t # # t ## # # t # # # # # t # A C B 1 A1 R1 R9 X6 X5 other Tri Hexa Voucher ## # # ## # ## T T T # # # ## t # # # ## ### ### # # ## H,P,T ### ## ## ### ## ## # # # ## # # ### ### # ## ### # # # ## ## ### H T T # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # UCoH BAa UCo Kew UCo BAa BAa AaUH UBCH UBCH BAa UCo BAa UCo UCo UCo UCo UCoH UCo UCo UCo BAa BAa UCo Aus UCo Den UCo BC BAa AaUH Den UCo BAa UCo UCo Kew UCo BAa UCo BAa UCo UCo BAa BAa UCoH Aus UCo UCo AaUH J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 239 Table 3*continued R Ziziphora capitata L. Z. tenuior L. IV.2. Lavanduleae Lavandula lanata Boiss. L. minutolii Bolle L. mulitixda L. L. mulitixda L. &canariensis' L. pedunculata Cav. L. pinnata L. "l &Buchii' L. stoechas L. L. stoechas v. leuchantha A C B 1 A1 R1 R9 X6 X5 other Tri Hexa Voucher # t # # # # # # IV.3. Elsholtzieae Collinsonia canadensis L. # Elsholtzia ciliata Thunb. E. densa Beit. # Mosla dianthera (Buch.-Ham.) Maxim. # Perilla frutescens (L.) Britt. # Perillula reptans Maxim. IV.4. Ocimeae Aeollanthus nyassae GuK rke ## A. stormsii GuK rke # Anisochilus carnosus Wall. Asterohyptis stellulata (Benth.) Epl. Catopheria chiapensis Gray ex Benth. # Coleus arabicus Benth. # C. chinensis C. pumilus Blanco Eriope hypeniodes Mart. ex Benth. ## Hyptis brevipes Poit. t H. capitata Jacq. t H. yoribunda Briq. # H. fruticosa Salzm. ex Benth. t H. lanceolata Poir. ## H. propinova # H. umbrosa Salzm. ex Benth. # Marsypianthes chamaedrys Kuntze Mesona procumbens Hemsl. Ocimum basilicum L. # O. basilicum **Ocim dark red++ # O. carnosum Link & Otto ex Benth. # O. gratissimum L. # O. selloi Benth. # Peltodon radicans Pohl ## Plectranthus fruticosus L'Herit ## P. glaucocalyx Maxim. ## P. parviyorus R.Br. # Pychnostachys meyeri GuK rke # t # # # ## # # # UCo UCo # # # # # # # # # UCoH UCoH UCo UCoH UCoH UCoH UCoH UBC # # # # # # UCo BAa UCo AaUH BAa AaUH # # # # # # # # # # # # # # # # # # # # # # # # # # # # Kew Kew BCoH Kew Kew BAaH BAa BAa Kew BCo BCo Kew Kew Kew Kew Kew AaUH AaUH Den UBC BCo Kew Kew Kew BAa UCo UCoH AaUH # # # ## ## # # ## ## T # # K K # ## # ## ## ## # # # # # ### ## # K *continued 240 J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 Table 3*continued R A P. coerulea Hook. Rabdosia japonica (Burman f.) Hara Rhaphiodon echinus Schauer V. Pogostemonoideae Anisomeles indica (L.) O. Kuntze Comanthosphace sublaneolata Moore Leucosceptrum canum Smith Pogostemon plectrantmoides Desf. VI. Scutellarioideae tr Holmskioldia humberti Moldenke H. sanguinea Retz. H. tettensis (Klotzsch) Vatke Salazaria mexicana Torr. Scutellaria alpina L. S. altissima L. S. columnea All. S. galericulata L. S. laterifolia L. S. peregrina L. C B 1 A1 R1 R9 X6 X5 other Tri Hexa Voucher # # t # # # K # t # # # # t # # # # # # # VII. Teucrioideae tr Aegiphila amazonica Moldenke # A. bogotensis (Spreng.) Moldenke A. brachiata Schlecht. A. cephalophora Standl. # A. costaricensis Moldenke # A. elata Sw. A. ferruginea Hayek & Spruce # A. yuminensis Vell. A. xlipes Mart. & Schau. A. glandulifera Moldenke A. hassleri Briq. A. intergrifolia (Jacq.) Jacks. A. laeta H.B.K. A. lanata Moldenke ## A. martinicensis Jacq. # A. mollis H.B.K. # A. panamensis Moldenke A. paraguariensis Briq. ## A. sellowiana Cham. ## A. verticilliata Vell. tr Amasonia arborea H.B.K. A. campestris (Aubl.) Moldenke A. hirta Benth tr Amethystea coerulea L. tr Caryopteris xclandonensis Rehder # C. divaricata (Sieb. & Zucc.) Maxim. C. incana (Thunb.) Miq. # C. paniculata C.B. Clarke # # # # # # # # ### ### # # # AaUH AaUH AaUH # # # # AaUH UBCH AaUH Kew # # # # # # # # # # AaUH AaUH AaUH AaUH BAa UCo UCo Den UCo BAa # # # # # # # # # # # # # # # # # # # # # # # # # # # # AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH UCo UCo AaUH BAa AaUH J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 241 Table 3*continued R C. tangutica Maxim. tr Clerodendrum angolense GuK rke C. bunchanani v. fallax (Lindl.) Bakh. C. bungei Steud. C. buruanum f. lindawianum (Lauterb.) Bakh. C. capitatum Hook. C. colebrokianum Walp. C. cyrtophyllum Turcz. C. deyexum Wall. C. discolor Vatke C. disparifolium Blume C. yoribundum R. Br. C. fortunatum L. C. fuscum GuK rke C. godefroyi Kuntze C. indicum Kuntze C. inerme (L.) Gaertner C. infortunatum L. C. kaempferi Fisch. ex Morr. C. laevifolium Blume C. laevifolium v. yetcheri Moldenke C. ligustrinum R. Br C. molle H.B.K. C. molle v. glabrescens Svenson C. myricoides v. niansanum Thomas C. nutans Jack C. paniculatum L. C. phillippinum Schau. C. phillippinum v. simplex Moldenke C. rehmannii GuK rke C. schmidtii C.B. Clarke C. schweinfurthii v. longitubum (De Wild.) Thomas C. serratum (L.) Spr. C. speciosissimum van Geert C. splendens G. Don C. strictum Baker C. thomsonae Balf. f. C. thomsonae Balf. f. C. trichotomum Thunb. C. trichotomum Thunb. C. ugandense Prain C. urticifolium Wall. C. villosum Blume C. viscosum v. helferi Moldenke tr Glossocarya mollis Wall. ex Gri!. G. siamensis Craib A ## C B 1 A1 R1 R9 X6 X5 other Tri Hexa Voucher # # # ## # # # # ## # ## ## ### # ## ## ## ## # # ### # # # # # UCo AaUH AaUH AaUH AaUH # # # # # # # # # # # # # # # BAa AaUH AaUH AaUH AaUH BAa AaUH AaUH AaUH AaUH AaUH UCo AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH # # # # # # # # # # # # # # AaUH BAa BAa AaUH BAa AaUH BAa AaUH BAa AaUH AaUH AaUH AaUH AaUH # # # # # # # # # # # # ## # # #! ## # ## # # # # # # ## ## ## ## ## # ### # P *continued 242 J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 Table 3*continued R A Isanthus brachiathus (L.) BSP tr Peronema canescens Jack. tr Tetraclea coulteri v. angustifolia (W. & S.) A. Nels. & Macbr. Teucrium bicolor Sm. T. capitatum L. T. chamaedrys L. T. yavum L. T. fruticans L. T. marum L. T. polium L. T. scorodonia L. t ## t C B 1 A1 R1 R9 X6 X5 other Tri Hexa Voucher # ### # # # # # # # # # # # ## # # VIII. Viticoideae tr Callicarpa acuminata H.B.K. C. americana L. # C. americana v. lactea F.J. Muller ## C. angustifolia King & Gamble C. arborea Roxb. ## C. candicans v. sumatrana (Miq.) ## Moldenke C. dichotoma (Lour.) K. Koch # C. ericlona Schau. C. giraldiana Hesse # C. japonica Thunb. ## C. longifolia f. yoccosa Schau. C. loueiri Hook. & Arn. ## C. macrophylla Vahl ## C. maingayiKing & Gamble # C. mollis Sieb. et Zucc. ## C. oshimensis v. iriomotensis (Masam.) ## Hatus. C. rubella Lindl. C. subpubescens Hook. & Arn. C. tomentosa (L.) Murr tr Cornutia coerulea (Jacq.) Moldenke ## C. grandifolia Schau. # C. microcalycina Pav. et Moldenke # C. odorata Poepp. ex Schau. ## C. pyramidata L. ## tr Geunsia farinosa Blume ## G. pentandra (Roxb.) Merr. ## tr Gmelina arborea Roxb. # G. arborea Roxb. G. asiatica L. G. asiatica v. villosa (L.) Bakh. # G. phillipensis Cham. ## tr Hymenopyramis acuminata Fletcher H. brachiata Wall. # # # # # # ## # # # # P ### ## # # ### ### ### ## # ## ## ### ### # # # # # UBCH AaUH AaUH # # # # # # # # UCoH UCoH BAa UCoH UCoH UCoH UCoH BAa # # # # # # AaUH AaUH UCo AaUH AaUH AaUH # # # # # # # # # # UCo AaUH AaUH BAa AaUH AaUH AaUH AaUH AaUH AaUH # # # # # # # # # # # # # # # # # AaUH AaUH AaUH UCo AaUH AaUH AaUH AaUH AaUH AaUH UBC AaUH AaUH BAa AaUH AaUH AaUH J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 243 Table 3*continued R A H. cana Craib H. parvifolia Moldenke H. siamensis Craib tr Petita domindgensis v. ekmani Moldenke tr Premna collinsae Craib P. cordifolia Roxb. P. foetida Reinw. ex Blume P. foetida Reinw. ex Blume P. fulva Craib P. gaudichaudii Schau. P. latifolia v. mollissima C.B. Ckarke P. microphylla Turcz. P. obtusifolia v. minor (Ridl.) Moldenke P. pyramidata Wall. P. racemosa Wall. P. tomentosa Willd. P. trichostoma Miq. P. venulosa Moldenke tr Teijsmanniodendron subspicatum Koord. C B 1 A1 R1 R9 X6 X5 other Tri Hexa Voucher # # # # # t # P # ### # # # # # # # # AaUH AaUH AaUH AaUH # # # # # # # # # # # # # # # AaUH AaUH BAa AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH Note: Species arranged according to classi"cation by Cantino, Harley and Wagsta! (1992). For the genus Vitex, see Table 4. Concentration estimates are indicated as: t: trace amount to low; #: low to medium; ##: high. The following compounds: A1, R1, R9, X6 and X5, are assinged: #, if observed; others are stated under `othera as H(hydroquinone), K (Ca!eic acid), P (protochatechuic acid) and T (thymohydroquinone). For explanation of symbols, see text. The following abbreviations are used, Tri " tricolpate and Hexa"hexacoplate pollen. tr in front of a genus means this genus is transferred from Verbenaceae. !Huang (1972) reported hexacolpate pollen in C. paniculatum, but described it as pericolpate, not homologous with that in Nepetoideae. extracts also containing the A-compound (Table 3). Finally, the artefact 6-hydroxycaffeic acid (R ) is frequently observed in the extracts from the present study. It is usually 9 observable from extracts containing compounds possessing ca!eoyl or dihydrocaffeoyl moieties. R, C and some A compounds ful"l this requirement. Another compound frequently present in Lamiaceae extracts gives rise to a characteristic spectrum at an elevated pH (See Fig. 2B). Due to the observed coupling constants we tentatively assign the spectrum as derived from b-hydroxy-(3,4,-dihydroxy-phenyl) ethanoid glycosides (b-alkoxy-) and designate the corresponding compound B . Because of the elevated pH applied we cannot exclude, B to be 1 1 a hydroxylation/degradation product of a genuine compound B in the extract. b-hydroxyacteoside (a B1 compound in our terminology) has been reported from related families. For example it was isolated from the dried fruit of Forsythia suspensa and of F. viridissima (Nishibe et al., 1982) together with acteoside, forsythiaside (forsythioside A) and suspensaside (forsythioside C). Acteoside and forsythiaside are 244 J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 both A compounds in the present terminology, and suspensaside is a B1 compound. We have applied the ESR technique to extracts of F. fortunii and F. viridissima and have in both cases observed the A spectrum. However, the B1 spectrum was observed only in F. fortunii, the lack of B1 in F. viridissima possibly being due to the fact that our experiment was performed on leaf extracts. Kvist and Pedersen (1986) tentatively assigned B1 as a hydroxylation product obtained from A compounds in the extracts. This assignment is questionable, as can be seen from Table 3, viz. many specimens do contain A in the extracts but do not furnish B1. From other extracts strong signals of B1 are observed, but A is lacking (cf. Clerodendrum rehmannii or Callicarpa rubella, Table 3). An interesting observation is that we never observe B1 in specimens containing rosmarinic acid apart from Teucrium scorodonica where A, R and B1 were observed together, a case which shall be discussed below. Among the artefacts we have observed in Reaction II we select two designated X5 and X6. They give rise to characteristic spectra and represent particular structures in the extracts. We use them as "ngerprints for pertinent compounds. X6 gives rise to an 8-line spectrum from three protons of the semiquinone nucleus. X5 yields a 2-line spectrum. It is known (Kvist and Pedersen, 1986) that 5,6,7-trihydroxy#avones (e.g. 4@ -methyl scutellarein) give rise to 2-line spectra from the single proton of the A-ring, with a splitting parameter nearly identical to the one of X5. We have previously observed X5 in 20 gesneriads, all con"ned to Cyrtandroideae (Kvist and Pedersen, 1986). Finally, when looking at Table 3 the presence/absence of a particular compound seems at places to follow an unpredictable pattern. Three explanations might be given: (a) seasonal variation or variations in growing conditions from specimen to specimen, (b) changes in the chemical contents in connection with the way the plant material has been treated prior to storage and "nally (c) compound deterioration due to long time storage. If we compare the results we have from experiments on fresh material with those obtained on herbal material we have observed the following: In a comprehensive study of 346 specimens of Verbenaceae (Sivebvk, 1982) the ubiquitous marker compound A was observed in 90% of freshly collected specimens (47 out of 52) and in only 50% of those obtained as herbal material (147 out of 294). For the Verbenaceae included in the present study the corresponding "gures are (19 out of 19, 100%) and (52 out of 117, 44%) for fresh and for herbal material, respectively. These results clearly point out that fresh material is preferable for chemotaxonomic work and that conclusions based solely on herbal material should be accepted with caution. Finally, we have excluded 32 specimens of Vitex in the above "gures because in none of the Vitex specimens did we observe A (see discussion below). 2.2. Taxonomic implications In the Lamiaceae classi"cation by Cantino et al. (1992), henceforth referred to as the CHW-classi"cation, which resulted as an updated outcome of the international symposium `Advances in Labiate Sciencea held at the Royal Botanic Gardens, Kew, 1991 the authors recognise eight subfamilies. J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 245 I. Ajugoideae, a small subfamily mainly based on morphological evidence as a segregate of Ajuga and a few other genera, II. Chloanthoideae, containing the genera formerly comprising Prostanthereae and Chloanthaceae, previously recognised as a separate family (or as a subfamily in Verbernaceae), III. Lamioideae, being the remains of Erdtman's, Lamioideae, since most genera have been distributed among six other subfamilies, IV. Nepetoideae, the largest subfamily and adopted from Erdtman's circumscription of subfamily Nepetoideae. It is the only subfamily further subdivided into tribes, V. Pogostemonoideae, here recognised as a subfamily not seen in Wunderlich's system, VI. Scutellarioideae, including the verbenaceous genus Holmskioldia and the genera Renskia and Tinnea from Ajugoideae, VII. Teucrioideae, comprised of many genera from the verbenaceous tribes Caryopterideae, Clerodendreae and Monochileae plus a few from Ajugoideae sensu Wunderlich (1967), VIII. Viticoideae, mostly comprised of the genera traditionally contained in the verbenaceous tribes Callicarpeae, Caryopterideae, Tectoneae, Teijsmanniodendreae and Viticeae. We have adopted this classi"cation and shall discuss each subfamily separately. The specimens studied and the compound distributions found are all collected in Table 3, apart from the genus Vitex, the result of which is shown in Table 4. The pollen morphological data is collected from the available literature (Cantino and Sanders, 1986; Huang, 1972; Wunderlich, 1967). I. Subfamily Ajugoideae: In this subfamily with 6 genera only three specimens of Ajuga have been investigated. All three contain A in line with the tricolpate pollen found in the subfamily. Neither chlorogenic acid (C) nor B1 has been observed but more specimens have to be investigated to judge whether this lack is a general feature for the subfamily. Both C and B1 are observed in the otherwise closely related Teucrium (see below). II. Subfamily Chloanthoideae: From this subfamily with 17 genera we have studied eight genera. Five contain A, again in accordance with the presence of tricolpate pollen (except in Prostanthera sect. Klanderia (F. Muell.) Benth., where it is hexacolpate) and three contain B1. The provisioned amalgamation of Prostanthereae and Chloanthaceae does not seem compromised. The lack of both A and B in the single 1 species of Hemigenia, Lachnostachys and Newcastelia is too limited a result to base "rm conclusions on. Pozhidaev (1992) found six colpi in the 2-celled Prostanthera coccinea. His result seems to rule out that the origin of three- and sixcolpate pollen grain in Lamiaceae is caused by a parallel transition from a 2-celled stage to a 3-celled one. It would be of interest to see whether the A compound is found in all Prostanthera no matter whether they are of the three- or hexacolpate type. III. Subfamily Lamioideae: From this large subfamily with about 55 genera we have investigated 21 genera. A has been found in 28 specimens out of 41 studied, again in 246 J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 Table 4 Occurrence of dihydroxyphenolics in taxa of <itex C Vitex agnus-castus L. V. agnus-castus L. V. altissima L.f. V. betsilensis ssp. barorum Humbert V. canescens Kurz V. capitata Vahl V. cofassus Reinw. ex Blume V. compressa Turcz. V. cymosa Bert. ex Spreng. V. doniana Sweet V. gamosepala Gri!. V. gigantea H.B.K. V. glabrata R.Br. V. leucoxylon L.f. V. limonifolia Wall. V. longisepala v. longibes Moldenke V. madiensis v. milanjiensis (Britt.) Pieper V. negundo L. V. negundo L. V. negundo v. heterophylla (Franch.) Rehd. V. orinocensis Kunth V. peduncularis Wall. ex Schauer V. pinnata L.f. V. polygama Cham. V. pseudo-negundo Hand.-Mazz V. quinata Druce V. rotundifolia L.f. V. siamica F.N. Williams V. triyora Vahl V. trifolia v. simplicifolia Cham. V. tripinnata (Lour.) Merrill V. vestita Wall. P R, # ## ## ## ## # # ## t ## ## # ## ## ## # ## TQ # # # ## # # ## ## # ## ## ## # ## ## # ## ## # # ## # # # t ## # Voucher BAa AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH UCo AaUH UCo AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH AaUH Concentration estimates for C (Chlorogenic acid), P (protochatechuic acid), R9 and TQ (a-tocopherolhdroquinone) are indicated as: t: trace amount to low; #: low to medium; ##: high. line with tricolpate pollen for the subfamily. Compounds with the A -structure, e.g. acteoside and lavandulifolioside have previously been isolated from Stachys lavandulifolia (Basaran et al., 1988), S. macrantha (Calis et al., 1992a) and from Leonurus glaucescens (Calis et al., 1992b). S. sieboldii (Nishimura et al., 1991) contains acteoside, isoacteoside, deca!eoylacteoside and stachyosides A and B, all of which give spectra similar to A. We have found A in all "ve species of Phlomis in line with the "nding of acteoside and phlinosides A, B and C from Phlomis linearis (Calis et al., 1990). B was found in 4 out of 5 species of Phlomis and chlorogenic acid (C) in all 1 three Lamium species studied B and C are of rare occurrence elsewhere in the 1 J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 247 subfamily. Hydroquinone (H) was observed in Stenogyne purpurea and protochatechuic acid (P) in Paraphlomis grasilis. The 2-line spectrum X was observed in 5 Marrubium astracanicum and in Phlomis russeliana. No phenolics were observed from two species of Leucas and one of Panzeria all three of which were obtained as herbal material. IV. Subfamily Nepetoideae: This subfamily is subdivided into tribes in the CHWclassi"cation viz. (a) Mentheae, as circumscribed by Briquet and Wunderlich and here greatly expanded by including Glechoneae, Hormineae, Lepechinieae, Meriandreae, Monardeae, Nepeteae, Prunelleae, Rosmarineae and Salvieae, (b) Elsholtzieae, already recognised as a group by Bentham, and "nally (c) Lavanduleae and (d) Ocimeae as circumscribed by Wunderlich (1967). All members of the subfamily have hexacolpate pollen and it has been amply asserted in the past that a corresponding taxonomic marker compound is rosmarinic acid. Harborne (1966) found the acid in 13 genera and noticed its absence from genera outside Nepetoideae, viz, Phlomis, Prostanthera, Scutellaria, Stachys and Teucrium. In Salvia it was absent in two out of seven genera, there replaced by chlorogenic acid. This is in line with our "nding shown in Table 3, where absence of rosmarinic acid often is compensated for by the presence of chlorogenic acid. Tribe Mentheae is the largest one in the subfamily with 72 genera. We have investigated 34 genera and observed rosmarinic acid (R) in 90% of the studied taxa (73 out of 81 species). Strong signals of R were observed in three Prunella vulgaris specimens collected at three di!erent localities. Hydroquinone (H) has been observed in Dracocephalum ruyschiana, Origanum vulgaris and Salvia hispanica. Hydroquinone has previously been isolated from O. majorana (Assaf et al., 1987). We have observed thymohydroquinone (T) in O. vulgaris, Satureja montana, S. thymbra and in all three studied taxa of Monarda. Thymoquinone was actually isolated from Monarda xstulosa more than 100 y ago (Liebermann and Iljiuski, 1885) and more recently from Nepeta leucophylla (Gupta et al., 1964). Neither A nor B has been observed in the tribe; a new 1 compound X , however, is observed in 24 species, e.g. in all three taxa of Monardella 6 studied. Tribe Lavanduleae with Lavandula as the only genus containing R in 6 of 8 studied species. The only other compound observed in this tribe was chlorogenic acid (C), actually replacing R in L. minutolii and L. multixda. Tribe Elsholtzieae with 6 genera contains R in 4 of 5 studied taxa. The absence of R in Elsholtzia ciliata as in Lavanduleae is compensated for by the presence of cholorogenic acid. Aritomi et al. (1985) isolated rosmarinic acid from Perilla frutescens v. acuta. We have observed thymohydroquinone (T) in Mosla dianthera. As with Lavanduleae X6 was not observed in the tribe. Tribe Ocimeae with 52 genera exhibited rosmarinic acid content in 25 species out of 30 studied (15 genera). X6 has been observed in Hyptis brevipes, Plectranthus fruticosus, P. glaucocalyx, Marcypianthes chamaedrys and Ocimum basilicum. Sumaryono et al. (1991) isolated rosmarinic acid from Orthosiphon aristatus, a genus not included in the present study, as well as free ca!eic acid. Their result is of interest since we have observed the ESR spectrum of free ca!eic acid (designated K in Table 3) in Anisochilus carnosus, Coleus arabicus, Plectranthus parviyorus and in Rhaphiodon echinus all four 248 J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 belonging to Ocimeae. However, these are the only species of Lamiaceae in which we have observed free ca!eic acid. It has often been discussed in the literature whether free ca!eic acid exists in plants. Many reports of its observation in the past are undoubtedly incorrect, the acid being an artefact liberated from ca!eoyl containing molecules in the isolation process. However, there is no doubt that we here encounter four extracts containing free ca!eic acid, and it seems likely that the free acid must have been present in the plants. If ca!eic acid is liberated in our extraction procedure or in the subsequent oxidation reaction we should observe the ESR spectrum of the free acid much more frequently. It was not observed in 590 specimens of Gesneriaceae (Kvist and Pedersen, 1986) or in 378 specimens of Verbenaceae (Sivebvk, 1982) and we have in the present study observed the spectrum only in the four species mentioned above. It was observed in 16 Lycopodium specimens out of 170 studied (Pedersen and "lgaard, 1982). In summary, Nepetoideae shows rosmarinic acid content in 110 species out of 127 studied (86%). The absence of rosmarinic acid in Anisochilus, Asterohyptis, Clinopodium, Hyssopus, Lallemantia, Marcypianthes, Mesona and Perillula might in each case be due to absence in the particular specimens we have studied. Thus, Litvinenko et al. (1975) and Zoz and Litvinenko (1979) observed the acid in Clinopodium and Hyssopus and reported presence as well as absence in specimens of Dracocephalum, Elsholtzia, Lallemantia, Nepeta and Salvia in line with our "nding. Furthermore, rosmarinic acid was reported from Amaracus, Arischrada, Hymenocrater, Kudrjaschevia, Lophanthus, Majorana and Schizonepeta (Litvinenko et al., 1975; Zoz and Litvinenko, 1979), Iboza (Harborne, 1966), and Orthosiphon (Sumaryono et al., 1991), all genera not included in the present study. Accordingly, rosmarinic acid has thus been reported from more than 60 genera of subfamily Nepetoideae. Neither A nor B was found in any of the 55 genera included in the present study. X was 1 6 observed in 29 species and seems con"ned to Nepetoideae. V. Subfamily Pogostemonoideae: In this subfamily with 7 genera we have studied four genera. We found A in all four in line with tricolpate pollen for the subfamily. Acteoside has previously been isolated from Leucosceptrum japonicum (Miyase et al., 1982). We have observed B in Anisomeles and Comanthosphace, and X5 in Comanthos1 phace and Leucosceptrum. VI. Subfamily Scutellarioideae: We have studied two of the four genera in this subfamily, viz, three species of Holmskioldia and seven of Scutellaria (Salazaria " Scutellaria). A was observed in Holmskioldia tettensis and Scutellaria galericulata only, and no other compounds were observed from any of the 10 species studied by the ESR technique, apart from X5 observed in two species. We believe X5 derives from baicalein or scutellarein, as both compounds occur frequently in Scutellaria species. They both give rise to 2-line spectra. The few phenolics observed in the subfamily is a fairly marked result and may be of taxonomic interest. VII. Subfamily Teucrioideae: This fairly large subfamily with 23 genera is a conglomerate of Rubiteucris and Schnabelia and 18 genera transferred from Verbenaceae plus Tetraclea, Teucrium and Trichostema from Ajugoideae sensu Wunderlich (1967). Ten genera were included in the present study. A and B1 exhibit a rather scattered occurrence, which might be a result of the use of mainly herbal material. It is thus of J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 249 interest to see that A is always found in specimens of fresh plant material, apart from in Amethystea coerula. A was observed in 40 species of the 83 studied and B in 21 1 species. In Clerodendron rehmannii B was observed and A was absent. No phenolics 1 were observed in any of three Amasonia species and in Amethystea coerulea. It has been questioned whether Ametystea is best assigned to Lamiaceae or some other family (Cantino and Sanders, 1986). The lack of phenolics in this species and in the three species of Amasonia might indicate a questionable assignment for these genera. Rosmarinic acid (R), which so far has been con"ned solely to subfamily Nepetoideae as seen above, showed up in Aegiphila mollis with A lacking. In Teucrium scorodonia A and B were detected as well as R. The simultaneous presence of R and 1 A in one plant has to our knowledge only been reported once before. Thus, De Tommasi et al. (1991) isolated rosmarinic acid and verbascoside (acteoside) from Momordica balsamina (Curcurbitaceae). The apparent break here of the otherwise ubiquitous observation that A and R are mutually exclusive is of particular interest. From an investigation of material of Teucrium Nabli (1976) suggested a trend from the tricolpate type through a number of transition types to a clearly hexacolpate type. We have reinvestigated the pollen type for T. scorodonia as well as A. mollis and found both posses tricolpate pollen. Gross et al. (1988) isolated teucrioside (A) from Teucrium chamaedrys. Finally, X is 5 here observed more frequently than in any of the other subfamilies studied. It is observed in 14 species from four genera all formerly belonging to Verbenaceae. VIII. Subfamily Viticoideae: This subfamily consists solely of genera (15) transferred from Verbenaceae. Our study includes 8 genera with 80 species. A and B are observed 1 with strong signals in most specimens of Callicarpa and Geunsia ("Callicarpa, the name retained as stated by collector). It should be noticed that absence in certain species only is seen for herbal materials. R is not observed from this subfamily having tricolpate pollen. One result is immediately striking, namely the total absence of A and B in any of the 30 species of Vitex. This unequivocal result clearly strengthens the 1 isolated position of Vitex. Another deviating result for this genus is the frequent observation of protochatechuic acid (P, 11 specimens) or chlrogenic acid (C, 9 specimens) the two compounds being mutually exclusive. P has a scarce occurrence elsewhere in the family (4 specimens). We have for practical reasons placed Vitex separately in Table 4. In concert with the result for Vitex the "ve species of Hymenopyramis are set o! by showing a total absence of A as well as B . Unexpected1 ly, however, rosmarinic acid was found in Hymenopyramis brachiate. A reinvestigation of the pollen for H. brachiate showed it to be of the tricolpate type. The placement of Hymenopyramis in Viticoideae might be questioned. Finally, the absence of A and B1 in Petitia domingensis v. ekmani and in Teijsmanniodendron subspicatum is of interest in relation to their transfer to Lamiaceae. More species of the two genera have to be investigated before a "rm conclusion can be made. However, the scattered occurrence of A in Premna as well as the nearly total absence of B1 (found only in Premna microphylla) does indicate that a chemotaxonomic di!erence exists between this genus and say Callicarpa. The scattered occurrence of A in Premna also seems in line with studies focusing on the isolation of A-type compounds. Thus, Otsuka et al. (1993) isolated verbascoside and premcoryoside, an 250 J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 iridoid glycoside conjugate of verbascoside (both being A-compounds in our de"nition) from P. corymbosa v. obtusifolia, whereas only ca!eoyl-containing compounds (C-compounds) were isolated from P. japonica and P. odorata (Otsuka et al., 1989). 3. Conclusion The present ESR-study on extracts from the leaves of 365 specimens representing 355 species and varieties of 110 genera of Lamiaceae has shown that two chemical characters give strong support to the subfamily division of the Lamiaceae as suggested by Erdtman (1945): Rosmarinic acid (R) con"ned to Nepetoideae and 3,4-dihydroxyphenylethanoid glycosides (A) con"ned to Lamioideae. Our results by and large support the most recent classi"cation of the Lamiaceae by Cantino, Harley and Wagsta! (CHW, 1992) the authors recognising eight subfamilies. Thus, rosmarinic acid (R) was observed in 110 species out of 127 studied and all con"ned to Nepetoideae sensu CHW (hexacolpate pollen). The observation of rosmarinic acid in 87% of the studied taxa of Nepetoideae strengthens this compound as a strong chemotaxonomic marker for the subfamily. 3,4-Dihydroxyphyenlethanoid glycosides (A) were observed in 111 species out of 228 studied and all con"ned to subfamilies with tricolpate pollen grains. 3,4-Dihydroxyphenylethanoid glycosides were unobserved from any of 5 Hymenopyramis or from any of 30 species of Vitex both genera belonging to Viticoideae sensu CHW. This result thus questions the transfer of these genera to Lamiaceae. In the case of Vitex the result not only deviates by the absence of A but by the frequent observation of chlorogenic acid (9 species) and of protochatechuic acid (11 species), the two compounds being mutually exclusive in the 32 specimens studied. The family relationship of Vitex is hereby questioned. The less frequent observation of A (found in 55% of the studied taxa, excluding the result for Vitex) than of R in Lamioideae and Nepetoideae sensu Erdtman, respectively, might be a result of the use of mainly herbal material in the former case, especially for genera transferred from Verbenaceae. Thus, A was observed in all 19 freshly collected specimens of verbenaceous genera, but only in 52 specimens out of 117 (44%) obtained as herbal material. R and A are shown to be mutually exclusive in Lamiaceae, apart from in one species, Teucrium scorodonia. Furthermore, rosmarinic acid was observed outside Nepetoideae in two more cases, viz. in Aegiphila mollis and in Hymenopyramis brachiata, where R occurred alone. These cases are of particular interest in discussion of trends in transitions leading from tricolpate through intermediate types to clearly hexacolpate pollen. A structure B , tentatively assigned as a b-hydroxy-(3,4-dihydroxyphenyl) ethanoid 1 glycoside, was observed in 49 species, solely con"ned to subfamily Lamioideae sensu Erdtman. It was found absent from Ajugoideae, Scutellarioideae and Nepetoideae sensu CHW. Hydroquinone occurred scattered in the family (4 species) and thymohydroquinone was observed in 7 species all belonging to Nepetoideae sensu CHW (tribes Mentheae and Elsholtzieae). J.A. Pedersen / Biochemical Systematics and Ecology 28 (2000) 229}253 251 4. Material and experimental Both herbarium and fresh plant material were investigated. Voucher specimens are deposited at the Botanical Institute, University of Aarhus (AaU), Botanical Museum, University of Copenhagen (UCo), Department of Botany, University of British Columbia (UBC), the Royal Botanic Gardens Kew (Kew). In Tables 3 and 4 plant names are stated as written by the collector. Finally, a number of common species were "eld collected in Denmark (Den) and some in Austria (Aus) and in British Colombia (BC). Herbal plant material was extracted with EtOH}H O (4:1) and the extracts used as 2 obtained. Fresh plant material was extracted with EtOH. The semiquinone radicals were formed from o-or p-quinols by raising the pH and shaking the mixture in air (Pedersen and "lgaard, 1982). In general two experiments were run on each extract. Reaction I: Twenty ll of the extract was mixed with 5 ll 0.1}0.2 M NaOH and the mixture was shaken in air. 10 ll mixture in a capillary was introduced into the magnetic resonance cavity and ESR spectra recorded of 3,4-dihydroxyphenylethanoid glycosides (A), rosmarinic acid (R), chlorogenic/neochlorogenic acid (C), protochatechuic acid (P), hydroquinone (H) and thymohydroquinone (T), immediately or after few seconds. In case of rapid disappearance a spectrum was easily restored by turning the capillary upside down and letting an air bobble pass through the solution. The spectrum then reappeared immediately after reintroduction of the sample into the cavity. This process could be repeated several times, the number being dependent on the actual concentration in the sample. Reaction II: Twenty ll of the extract was mixed with 5 ll 1M NaOH and the mixture was shaken in air. The increased pH made hydrolysis and hydroxylation more rapid for the pertinent compounds, and spectra of the secondary products A1, R1, R9, X5, X6 and B1 (genuine?), eventually appeared after a delay of seconds to minutes (for an explanation of abbreviations used, see text). Acknowledgements The author thank Professor G.H.N. Towers for stimulating discussions, F.G. Herring for generously providing ESR equipment and Dr. R. Harley for provision of plant material. References Aritomi, M., Kumora, T., Kawasaki, T., 1985. 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