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
#
#
#
#
#
#
#
#
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#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
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.
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