Directory UMM :Data Elmu:jurnal:B:Biochemical Systematics and Ecology:Vol28.Issue10.Dec2000:
Biochemical Systematics and Ecology 28 (2000) 991}1007
Leaf #avonoids as systematic characters
in the genera Lavandula and Sabaudia
Tim M. Upson!,1, ReneH e J. Grayer", Jennifer R. Greenham!,
Christine A. Williams!,*, Farag Al-Ghamdi!, Fen-Hui Chen!
!Department of Botany, University of Reading, Whiteknights, Reading RG6 6AS, UK
"Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK
Received 28 September 1999; accepted 3 December 1999
Abstract
A comprehensive survey of the leaf #avonoids of the genus Lavandula and the related
Sabaudia group was carried out using two-dimensional paper chromatography and highperformance liquid chromatography. The #avonoid patterns obtained were found to be
systematically informative at the infrageneric level. Three main groupings were identi"ed: the
"rst containing sections Lavandula, Dentata and Stoechas characterised by the accumulation of
#avone 7-glycosides; the second containing sections Pterostoechas, Subnuda and Chaetostachys
characterised by the accumulation of 8-hydroxylated #avone 7- and 8-glycosides; the third
encompassing the Sabaudia group and accumulating both #avone and 8-hydroxylated #avone
7-glycosides. Such a grouping of taxa is congruent with data from other disciplines, although it
is not recognised in any present classi"cations. The taxonomic and evolutionary implications of
the #avonoid data are discussed. ( 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Lavandula; Sabaudia; Lavender; Lamiaceae; Flavonoids; Chemotaxonomy
1. Introduction
The genus Lavandula (lavender, Lamiaceae) is distributed from the Canary and
Cape Verde Islands and Madeira, across the Mediterranean Basin, North Africa,
* Corresponding author. Tel.: #44-118-9-3181-68; fax: #44-118-9-7536-76.
E-mail address: christine.williams@reading.ac.uk (C.A. Williams)
1 Present address: Cambridge University Botanic Garden, Cory Lodge, Bateman Street, Cambridge CB2
1JF, UK.
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 1 3 - 2
992
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
South}West Asia, the Arabian Peninsula and tropical NE Africa with a disjunction to
India. Although the name of the genus is familiar to many people, only a few species
are well known, e.g. L. angustifolia Miller or English lavender, L. latifolia or spike
lavender, and L. stoechas or French lavender. The English lavender is widely cultivated in gardens and is also an important essential oil crop from which many cultivars
have been selected.
The last revision covering the whole genus was undertaken more than 60 years ago
by Chaytor (1937), but more recently there have been a number of #oristic revisions
covering certain geographical areas, e.g. by Miller (1985) on the Arabian and NE
tropical African species. Based on Chaytor's work and that of others, six sections can
be recognized in Lavandula: Stoechas Ging. (3 spp.), Lavandula (3 spp. ), Dentata (1 sp.),
Pterostoechas (15 spp.), Subnuda (8 spp.) and Chaetostachys (2 spp.).
On the basis of its hexacolpate pollen (Erdtman, 1945) and accumulation of
essential oils (Hegnauer, 1989), Lavandula clearly belongs to the subfamily
Nepetoideae of the Lamiaceae. According to phylogenetic and molecular studies,
Lavandula is a distinctive clade in the Nepetoideae without close relatives (Cantino
et al., 1992; Kaufmann and Wink, 1994; Wagsta! et al., 1995). The nearest relative
appears to be the tribe Ocimeae. However, there is a small genus of two or three
species native to the Arabian Peninsula and NE tropical Africa, called Sabaudia,
which has sometimes been treated as part of Lavandula (Cufodontis, 1962), although
its tribal position within the Lamiaceae has not yet been fully established.
A vast literature exists on the essential oils in Lavandula, reviewed by Boelens
(1995). For the taxonomist the use of essential oils as characters for the classi"cation is
limited by inherent problems of natural variability, although at lower taxonomic
levels this can be used to help recognize cultivars (Grayer et al., 1996). The chemical
composition and ratios of the individual components making up the oils are also
known to change in response to environmental conditions, such as water and nutrient
stress or time of year (Ross and Sombrero, 1991). These inherent problems of variation
mean that other classes of chemical constituents such as #avonoids are often of greater
use and signi"cance to the systematist.
The utility of #avonoids within the Lamiaceae has been demonstrated at various
taxonomic levels, including the assessment of generic relationships within subfamilies,
generic a$nities of species, and even in verifying the parents of hybrids (reviewed by
TomaH s-BarberaH n and Gil, 1992). With regard to the genus Lavandula, no #avonoid
survey of the genus has yet been undertaken, although some species have been
investigated as part of wider taxonomic surveys of the family. The following
#avonoids were reported from L. dentata (Ferreres et al., 1986): genkwanin (apigenin
7-methyl ether), luteolin, apigenin, luteolin 7-glucoside, apigenin 7-glucoside, luteolin
7-rutinoside, vitexin and vicenin-2. Several species of Lavandula were investigated as
part of a survey on the occurrence of 6-hydroxy-, 6-methoxy- and 8-hydroxy#avone
glycosides within the Lamiaceae and related families (TomaH s-BarberaH n et al., 1988),
but no total #avonoid pro"les were given. The major #avonoids in L. stoechas were
apigenin 7-glucoside, luteolin, luteolin 7-glucoside and luteolin 7-glucuronide (Xaver
and Andary, 1988). Very recently, a number of #avone glycosides with extra hydroxylation in the 8-position were reported from L. pubescens and L. coronopifolia,
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
993
species both belonging to section Pterostoechas. These compounds were the 7glucoside and 8-glucuronide of hypolaetin, and the 8-glucuronides of hypolaetin
4@-methyl ether and isoscutellarein (El Garf et al., 1999). Glycosides of 8-hydroxyflavones are uncommon plant constituents, especially the 8-glycosides, and in several
cases have provided useful characters at the sectional level within genera of the
Lamiaceae (TomaH s-BarberaH n et al., 1988; TomaH s-BarberaH n and Gil, 1992). To see
whether that is also the case in the genus Lavandula, we have surveyed a large
proportion of the species of the genus for the presence or absence of these and other
#avonoids, and also studied representatives of Sabaudia in the hope that #avonoid
pro"les would help in clarifying relationships between this genus and Lavandula.
2. Materials and methods
2.1. Plant material
Specimens and voucher details for the taxa surveyed are presented in Table 1. The
fresh material used in the investigations was "rst grown in a greenhouse and then
moved outside to grow under natural daylight for six weeks before extraction.
2.2. Extraction
A few chopped leaves of each sample were placed in a test tube, covered with, ca.
5 ml of 100% MeOH for fresh material or 70% aqueous MeOH for dry herbarium
material, and placed in a heating block at 703C for about 5 min. This procedure kills
the plant tissue preventing enzymic oxidation or hydrolysis. The samples were left to
extract for at least 8 h or overnight. Excess chlorophyll was removed using petroleum
ether (BP 40}603) before the extracts were evaporated to dryness on watchglasses.
2.3. Two-dimensional (2-D) -paper chromatography
The dried extracts were reconstituted using 80% MeOH and run two-dimensionally on quarter sheets of Whatman no. 1 chromatography paper. The chromatograms were run by descent in BAW (n-BuOH, HOAc, H O"4 : 1 : 5; v/v; upper
2
layer) "rst direction, and in 15% aqueous HOAc or H O in the second direction.
2
After drying, the chromatograms were viewed in UV light (366 nm) "rst without and
later in the presence of ammonia vapour. The colours of the spots before and after
NH were recorded and R -values calculated.
3
&
2.4. Paper electrophoresis
Extracts were applied as spots along a central axis on a sheet of Whatman no.
3 chromatography paper, measuring 19]28.5 cm. A spot of rutin (quercetin 3rutinoside) was used as a negative control, and quercetin 3-glucuronide as a positive
Table 1
Specimen and voucher details for the taxa surveyed. Accession numbers are given for those living specimens held in the collection at the University of Reading;
herbarium acronyms refer to vouchers and follow Holmgren, Keuken and Scho"eld (1981). Authority abbreviations follow Brummitt and Powell (1992).
L"living material used, H"herbarium material used
994
Taxa
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
Specimen and voucher details
Section Lavandula
L. angustifolia Mill. subsp. angustifolia
L
L. angustifolia Mill. subsp. pyrenaica (DC.) Masclans
L
L. angustifolia Mill. subsp. delphinensis (Jord.)
O. Bolòs & Vigo
L. latifolia Medic.
L
L. lanata Boiss.
L
Section Stoechas
L. stoechas L. subsp. stoechas
L
L. stoechas L. subsp. atlantica Braun-Blanq
L
L. stoechas L. subsp. cariensis (Boiss.) Rozeira
L. stoechas L. subsp. luisieri (Rozeira) Rozeira
L
L
L
Cultivated ex. Kiev Botanic Garden, Russia (RNG); Cultivated ex. Tsukuba Medicinal
Plant Research Station, Japan (RNG); Cultivated variant (RNG)
Cultivated ex. Botanischer Garten Kiel (RNG); Spain, Prov. of Huesca, Pyrenaen PlanBenasque ex. Botanischer Garten Berlin Dahlem (RNG); France, Frankreich Pyrenees
Orientalis ex. Botanischer Garten Berlin Dahlem (RNG)
Cultivated ex. Bundesgarten Wien } Alpengarten Im Belvedere (RNG)
France, Prov. of Aude, Montagne de Tauch ex. Jardin Botanique Leige (RNG); France,
Aude Fore( t de Rives-Hautes ex. Leige (RNG); Spain, Prov. Gerona Angles, ex. Botanischer Garten Berlin Dahlem (RNG); Italy, Siena, Pomona in Chianti ex. Jardin Botanico
Siena (RNG); France Angers ex. Instituto Botanico Siena (RNG)
Spain, Prov. of Granada, Sierra Nevada ex. Botanischer Garten Berlin Dahlem (RNG);
Cultivated ex. Jardin Botanique Ronen (RNG); Spain, Prov. of Malaga, S.L. Jury 13138
(RNG)
Corsica, Mt. Tomboni, L. Springate 93.289 (RNG) Crete, J.D. Ross s.n. (RNG); Turkey,
Mugla province, SW of Marmaris, L.C. Jury 334 (RNG); France, Angers ex. Jardin
Botanique de Dijon (RNG); Spain, Prov. of Granada, Sierra Nevada ex. Jardin Botanico
Cordoba (RNG); Morocco, SE of Tetouan, 1 km N of Oued Laou, S.L. Jury 12408 et al.
(RNG)
Morocco, Rif Central, 17 km S. Ketama, S.L. Jury 11566 et al. (RNG); Morocco, Haut
Ouerrha, 47 km S. of Ketama, S.L. Jury 11575 et al. (RNG)
NW Turkey, Balikesir, Haziren s.n. (RNG)
Portugal ex. Jardim Botanico Porto (RNG); Portugal, Algarve, Serra do Caldeira8 o ex.
Jardim Botanico Lisboa (RNG) Spain, Sa de CoH rdoba ex. Jardin Botanico Cordoba (RNG);
Portugal, Coimbra ex. Jardim Botanico Coimbra (RNG)
L
L. stoechas L. subsp. pedunculata (Mill.) Samp.
ex. Rozeira
L. stoechas L. subsp. sampaiana Rozeira
L
L
L. viridis L@HeH r.
L
Section Dentata
L. dentata L. var. dentata
L
L. dentata L. var. candicans Batt.
L
Section Pterostoechas
L. antineae Maire
H
L. buchii Webb & Berthel. var. buchii
H
L. buchii Webb & Berthel. var. gracile LeoH n
L
L. canariensis Mill.
L
L. coronopifolia Poir.
H
L
L. mairei Humbert var. mairei
L
L. mairei Humbert var. antiatlantica Maire
H
L
Portugal, Prov. Baixo Alentejo, Reguengos de Monsaraz ex. Jardim Botanico de Lisboa
(RNG)
Spain, Prov. of Leòn, La Magdalena ex. Jardin Botanico Bilbao (RNG); Spain, Prov. of
Avila, Sierra de Gredos ex. Botanisher Garten OsnabruK ck (RNG)
Portugal, Prov. Alto Alentejo, Marva8 o ex. Jardim Botanico Lisboa (RNG) Portugal, nr.
Coimbra ex. Jardim Botanico Coimbra (RNG)
Portugal, outside Barrada ex Joan Head 91.10 (RNG); Portugal, Barranco Velho ex. Joan
Head 91.08 (RNG); Cultivated ex Jardin Botanico Nice (RNG)
Balearic Islands, Mallorca (RNG); Morocco, S. of Tetouan on rd. to Oued Laou, Cap
Mazari, S.L. Jury 12383 et al. (RNG); Morocco, N. of Agadir, rd. to Immouzzer, S.L. Jury
11984 et al. (RNG); Cultivated ex. Jardin Botanico Barcelona (RNG)
Morocco, NW of Agadir, Cap Rhir, S.L. Jury 12000 et al. (RNG); Morocco, nr. Al
Hoceima, S.L. Jury 13533 (RNG)
Algeria, Hoggar Mts, Miller 4144 et al. (E); Algeria, Hoggar Massif, Oued Ideles, D.
Podlech 36932 (M)
Canary Islands, Tenerife: Anaga Peninsula, near Las Carboneras, T.M. Upson 299 (RNG);
Anaga Peninsula, NW of Taganana, Playa de Benijo, T.M. Upson 307 (RNG)
Canary Islands, Tenerife, Teno Region ex. Joan Head (RNG); Teno Region, c. 6 km SW of
Tamaimo, T.M. Upson 301 (RNG); Teno Region, Mirador de Don Pompeyo, nr.
Buenavista, T.M. Upson 297 (RNG)
Cultivated ex. Norfolk Lavender (RNG); Canary Islands, Gran Canaria, Arucas, Joan
Head 93.21 (RNG)
Canary Islands, Tenerife, above San Juan, T.M. Upson 298 (RNG)
Morocco: SW Anti-Atlas, c.27 W of Tata, S.L. Jury 14464 et al. (RNG); SW Anti-Atlas, c.
20 km W of Tata, S.L. Jury 14448 et al. (RNG)
Morocco: 24 km N. of Er Rachidia, S.L. Jury 14612 (RNG); c. 26 km ESE of Tinerhir, S.L.
Jury 14589 (RNG)
Morocco, Gorges du Ziz, S.L. Jury 14614 (RNG)
Morocco, SW Anti-Atlas: W of Tata, S.L. Jury 14463a et al. (RNG); 27 km NE of
Tafraoute, S.L. Jury 14384 et al. (RNG); SE of AmK t-Baha, S.L. Jury 14385 et al. (RNG)
995
2continued
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
L. stoechas L. subsp. lusitanica (Chaytor) Rozeira
996
Table*continued
Taxa
Specimen and voucher details
L
L. minutolii Bolle var. minutolii
L
L. minutolii Bolle var. tenuipinna Svent.
L. multixda L.
L. pinnata L. ff.
H
L
Morocco: Gorges du Ziz, S.L. Jury 14615 (RNG); Morocco: Immouzzer Valley, 10 km
N of Agadir, S.L. Jury 14245 (RNG); Morocco: High Atlas, c. 12 km NNE of Ijoukak, S.L.
Jury 14200 (RNG)
Canary Islands, Gran Canaria, Joan Head s.n. (RNG); Canary Islands, Gran Canaria,
Roque Bentriga ex. Botanischer Garten Berlin Dahlem (RNG)
Canary Islands, Tenerife, Teno Region, near Masca, T.M. Upson 295 (RNG)
Culivated ex. Jardin Botanique De Dijon, France (RNG)
Cultivated ex. Orto Botanico Torino (RNG); Cultivated ex. Botanischer Garten Innsbruck
(RNG)
Cape Verde Island ex. Botanischer Garten Bonn (RNG)
Cape Verde Islands, mountains SW of the village Ribeira Grande, below Faja8 Bedonda,
26.1.1982, C. Brochmann 533/82 (O)
High Atlas, Toubkal range, side of gorge from refuge Neltner to Imlil village, 14.7.1966,
T.K. Thorp no. 95 (BM)
L. rotundifolia Benth.
L
H
L. tenuisecta Coss. ex. Ball
H
Section Subnuda
L. aristibracteata A.G. Mill.
L. dhofarensis A.G. Mill. subsp. dhofarensis
L. nimmoi Benth.
L
L
H
L. subnuda Benth.
L
Section Chaetostachys
L. bipinnata Kuntze
L
India, Prov. of Maharashtra, Poona District, Vede-Bowdhan ex. Botanical Survey of India
(BSI, RNG)
Sabaudia group
S. atriplicifolia (Benth.) Chiov.
S. erythreae Chiov.
H
H
Yemen Arab Republic, Kaukaban, J.R.I. Wood Y/75/311 (BM)
Ethiopia (Eritrea), Monti Lesa, A. Pappi (C)
Somalia, Surundi Hills, M. Thulin 9051 (RNG)
Oman, Dhofar, Wadi Sahalnawt, nr. Salalah, A.G. Miller 6158 (RNG)
Socotra, base of escarpment c. 6 km SE of Qalansiyah, 23.2.1989, A.G. Miller et al. 8395
(E)
N. Oman, Wadi Mistral, S. Gbazdrfai 2482 (RNG); Oman, Musandam by road to Khawr
Niad, H.D.V. Prendergast (RNG)
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
L. maroccana Murb.
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
997
control. The paper was subjected to electrophoresis at pH 4.4, using a sodium
acetate/acetic acid bu!er at 400 V for about 2 h.
2.5. Preparative paper chromatgraphy (PPC)
Flavonoids were separated from rosmarinic acid and other ca!eic acid derivatives
which interfered with the HPLC analysis of crude extracts by means of PPC. Each
crude extract was applied as a streak onto one quarter sheet of Whatman no.
3 chromatography paper. This was run by descent in 15% HOAc. The bottom half of
the paper which showed dark #avonoid bands in UV light (roughly from the origin to,
ca. R 0.50) was cut out, cut into 2 cm squares, and these were eluted in 80% MeOH
&
for 3}4 h. The top half of the paper (R -value above 0.50) which showed #uorescent
&
blue bands of ca!eic acid derivatives such as rosmarinic acid (R 0.58 in 15% HOAc)
&
was not eluted. The #avonoid eluate was evaporated and resuspended in ca. 0.8 ml of
80% MeOH. This was "ltered using a 13 mm Gelman Acrodisc, pore size 5 lm, ready
for HPLC analysis. A few extracts were further separated by PPC to isolate
#avonoids, using the solvent BAW (see above) as a second puri"cation step.
2.6. HPLC
The HPLC system consisted of a Waters 600E systems controller with a Waters 996
photodiode array detector. Injection of the extracts (40 ll) took place with a Waters
717 autosampler. Millennium software was used to record and analyse the results.
Two di!erent elution programmes were used; a LiChrospher 100RP-18 column was
employed for programme 1 and a Bondapak Phenyl RP-18 column for programme 2.
Both columns measured 4.0 mm (i.d.) ]250 mm with a 5 lm particle size. Elution
took place by means of two solvents, solvent A consisting of 2% of aqueous HOAc
and solvent B of MeOH, HOAc, H O, 18: 1: 1, and elution started with 25% of B at
2
t"0. For programme 1 there was a linear gradient reaching 100% B at t"20 min,
but for programme 2 it reached 65% B at t"23 min. In both programmes this was
followed by an isocratic elution with 100% B for 5 or 1 min, respectively, before going
back to the initial conditions. The #ow rate was 1.0 ml/min, and the temperature of
the column was 253C.
2.7. Identixcation of yavonoids
The #avonoids were identi"ed by comparing R -values on paper chromatograms,
&
colours of spots, HPLC R s and UV spectra of the compounds in the extracts with
5
those of standard markers. Puri"ed compounds were subjected to UV spectroscopy
using shift reagents to determine substitution patterns (Mabry et al., 1970), and to acid
hydrolysis to determine the aglycone and sugar moieties (Harborne, 1998). The
following #avonoid standards were available for comparison: luteolin 7-glucoside,
apigenin 7-glucoside, vitexin, apigenin, genkwanin (all purchased from Apin); luteolin
7-glucuronide, apigenin 7-glucuronide, (#avonoid collection at Reading University);
hypolaetin 8-glucuronide, its 4@-methyl ether, isoscutellarein 8-glucuronide,
998
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
hypolaetin 7-glucoside (#avonoid collection at Kew); xanthomicrol and salvigenin
(kindly provided to R.J.G. by Prof. E. Wollenweber).
3. Results
The extracts of 23 species of Lavandula and two of Sabaudia were examined by
means of 2-D paper chromatography. In addition, seven di!erent subspecies of L.
stoechas were studied together with three subspecies of L. angustifolia and two
varieties of L. dentata, L. buchii, L. mairei and L. minutolii. Besides, for many of the taxa
more than one accession was examined (see Table 1). Extracts of 13 species, representing all sections, were subjected to paper electrophoresis to check for the presence of
#avonoid glucuronides which are characteristic constituents of the family Lamiaceae
(TomaH s BarberaH n et al., 1988). The crude extracts used for the 2-D paper chromatograms were also analysed by HPLC. However, the HPLC traces did not give clear
results, because of the presence of large amounts of rosmarinic acid and related
substances in the extracts, which masked many of the #avonoid peaks and deformed
their UV spectra. For this reason about half of the extracts, representing all six
sections of Lavandula and all species of Sabaudia, were puri"ed by means of PPC to
separate the #avonoids from rosmarinic acid. HPLC of these puri"ed #avonoid
fractions gave much better results.
To identify some of the compounds, an extract of L. angustifolia ssp. pyrenaica was
used to isolate #avonoids by means of preparative paper chromatography and
preparative paper electrophoresis. Ten #avonoids were obtained, 1+8, 18 and 19.
Their colours on the chromatograms before and after fuming with ammonia, R &
values in two or three solvents, HPLC retention times in two di!erent solvent systems
and diode array UV spectra were determined (see Table 2). In addition, 1, 2, 4, 5, 7 and
8 were studied by UV spectroscopy using shift reagents (Mabry et al., 1970) and by
means of acid hydrolysis of the compounds and subsequent analysis of the aglycones
and sugars (Harborne, 1998). In this way, 1 and 2 were identi"ed as the 7-glucoside
and 7-glucuronide of luteolin, respectively, and 4 and 5 as apigenin 7-glucoside and
7-glucuronide. These identi"cations were con"rmed by means of co-chromatography
with standard markers on paper and by HPLC. After partial and total hydrolysis of
7 and 8 and analysis of the hydrolysis products, these compounds were tentatively
identi"ed as luteolin 7,4@-di-glucuronide and luteolin 7-glucoside-4@-glucuronide. No
standards were available for comparison. Not enough pure compound for 3 and 6 was
obtained for complete identi"cation. HPLC of these compounds revealed that 3 had
a similar UV spectrum to 1 and 2, but a longer retention time. The colour of 3 on
chromatograms in UV light after fuming with ammonia (yellow}green) and the UV
spectrum suggested that it may be a chrysoeriol 7-glycoside. In the same way, 6 may
be a glycoside of a methylated apigenin derivative. Compounds 18 and 19 were
identi"ed by means of co-chromatography with standards as the #avone aglycones,
apigenin and genkwanin.Table 2 shows another 11 #avonoids, 9}17, 20 and 21,
frequently encountered in extracts of Lavandula species, which were completely or
partly identi"ed by means of co-chromatography with standards using HPLC and
Table 2
Chromatographic and UV spectral data of #avonoids from Lavandula and Sabaudia species!
Compound
9
10
11
12
13
14
15
16
17
18
19
20
21
Colour
in UV
Luteolin 7-O-glucoside
D
Luteolin 7-O-glucuronide D
Chrysoeriol 7-O-glycosideH D
Apigenin 7-O-glucoside
D
Apigenin 7-O-glucuronide D
Derivative of 4 or 5
n.d.
Luteolin 7,4@-di-O-glucur.H D
Luteolin 7-O-glucosideD
4@-O-glucuronideH
6-OH-luteolin 7-OD
glycosideH
Scutellarein 7-O-glycosideH D
Vitexin
D
Hypolaetin 8-O-gluD
curonide
Derivative of 12
D
Hypolaetin 4@-methyl
D
ether 8-O-glucuronide
Isoscutellarein 8-O-glucur. D
Hypolaetin 7-O-glucoside D
Isoscutellarein 7-OD
glycoside
Apigenin
D
Genkwanin
D
Xanthomicrol
D
Salvigenin
D
R in BAW
&
R in 15%
&
HOAc
R in H O
&
2
R in solv.
5
1 (min)
R in solv.2
5
(min)
UV spectrum
(j
nm)
.!9
Y
Y
GnY
dYGn
dYGn
n.d.
Br
Br
0.40
0.32
0.42
0.58
0.50
n.d.
0.15
0.15
0.12
0.15
n.d
0.25
0.28
n.d.
0.40
0.40
0.02
0.09
n.d.
0.04
0.15
n.d.
0.69
0.69
14.2
14.1
15.9
15.7
15.7
17.2
n.d.
n.d.
17.4
17.4
21.0
20.3
20.3
23.7
12.8
12.8
256,
256,
252,
268,
268,
268,
269,
269,
D
0.25
0.08
n.d.
12.6
n.d.
285, 348
D
dY
D
0.35
0.40
0.40
0.13
0.30
0.12
n.d.
n.d
n.d.
14.1
13.2
15.9
n.d.
n.d.
n.d.
285, 338
269, 338
256sh, 271, 354
D
D
0.45
0.48
0.25
0.14
n.d.
n.d.
16.7
17.3
n.d.
n.d.
256sh, 271, 354
257, 271, 352
D
D
D
0.55
0.20
0.35
0.20
0.08
0.15
n.d.
n.d.
n.d.
17.4
13.9
15.4
n.d.
n.d.
n.d.
271, 343
276, 300sh, 343
276, 306, 328sh
Ygn
GnY
D
D
0.85
0.90
0.90
0.90
0.08
0.08
0.15
0.15
n.d.
n.d.
n.d.
n.d.
20.5
24.2
23.0
24.9
26.8
32.5
n.d.
n.d.
Colour
UV#NH
3
268,
268,
281,
276,
267sh, 350
267sh, 350
267sh, 348
338
338
338
336
336
338
338
290sh, 333
333
999
!Solv. 1 and Solv. 2: HPLC solvent programs 1 and 2, respectively. For composition of the solvents used for HPLC and PC, see Experimental.
Abbreviations: D"dark; Y"yellow; L"light; Gn"green; d"dull; Br"brown; n.d."not determined.
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
1
2
3
4
5
6
7
8
Identi"cation(H tentative)
1000
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
PC. The 8-hydroxylated #avonoids 12, 14}16 were previously isolated from two
species of Lavandula, L. coronopifolia and L. pubescens, and identi"ed by means of
NMR, MS and UV spectroscopy as the 8-glucuronides of hypolaetin, hypolaetin
4@-methyl ether and isoscutellarein and the 7-glucoside of hypolaetin, respectively (El
Garf et al., 1999). The compounds detected and identi"ed (sometimes tentatively)
during the combined HPLC, PC and electrophoresis analyses are presented in Table
3. The external #avonoids 18}21 (see Table 2) may be present in a larger quantity and
in more species than is shown in Table 3, as the methods of extraction and the HPLC
programmes were more suitable for #avonoid glycosides than for external #avones.
Absorbance of the peaks at the j
of each #avonoid was used as a measure of the
.!9
relative amounts (see Table 3). The solvent systems used for HPLC, which contained
a small percentage of HOAc, did not distinguish between glucosides and glucuronides
of the same aglycone, as these had more or less the same retention times. However, the
presence of both 1 and 2, and both 4 and 5 in all taxa investigated in sections Stoechas,
Dentata and Lavandula was con"rmed by means of paper electrophoresis of the
extracts and 2-D paper chromatography in BAW and water. Flavonoid glucuronides
are very mobile in water compared with the corresponding monoglucosides and so the
two are easily separated in this system.
A number of accessions were investigated by means of 2-D paper chromatography
only. As this technique is generally less sensitive than HPLC, only presence of the
major #avonoids 1, 2, 4 and 5 and the 8-hydroxy#avone glycosides 12 and 16 could be
scored. The latter compounds are easy to detect on paper chromatograms because
they do not change colour in UV light after fuming with ammonia. Moreover, they
have very characteristic UV spectra. These results are presented in Table 4 and largely
agree with the "ndings of the more detailed analyses presented in Table 3.
The #avonoid pro"les observed on the 2-D chromatograms and HPLC traces
showed very little variation among di!erent accessions, subspecies and varieties of the
same species. Moreover, there was surprisingly little variation in major #avonoid
pro"les among species belonging to the same section, any di!erences being mainly
quantitative. Section Lavandula appeared to be the most uniform group in that all
members produced #avone monoglycosides 1}6, #avone diglycosides 7 and 8, and the
aglycones, apigenin (18) and genkwanin (19). Again, in section Stoechas the di!erences
between the three taxa were very minor. However, in section Pterostoechas only two of
the seven taxa fully investigated (Table 3) had identical #avonoid pro"les, namely L.
maroccana and L. rotundifolia, although in all seven species 8-hydroxy#avone glycosides were the major #avonoid constituents. Lavandula mairei var. mairei di!ered from
all other members of this group in the presence of a chrysoeriol 7-glycoside (3) and
apigenin glycosides (4 and 5) and the absence of 8-hydroxy#avone glycosides 12}15.
Again, L. minutolii var. minutolii was distinguished by the absence of simple #avone
glycosides and the presence of #avone aglycones. Within section Subnudae, L. aristibracteata and L. subnuda have almost identical leaf #avonoid pro"les with the absence
of the simple #avone glycosides found in L. dhofarensis and L. nimmoi.
However, at sectional level profound di!erences were found in that the distribution
of the di!erent #avonoid classes, i.e. #avone glycosides, #avone diglycosides, 6hydroxy#avone glycosides, 8-hydroxy#avone glycosides and #avone C-glycosides,
Table 3
Presence of #avonoids in species, subspecies and varieties of Lavandula and Sabaudia based on HPLC, PC and electrophoresis results%
8-OH Flavone
8- glycosides
7/8
9
10
11
12
13
14
15
16
17
18
19
20
21
## ## ## $
## ! ## $
## ## ## #
!
!
!
!
!
!
!
!
!
$
$
$
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
$
$
$
$
$
$
!
!
!
!
$
$
L. dentata var. dentata%
Section Lavandula
#
!
#
$
!
$
## ## !
!
!
!
!
!
$
$
!
!
L. angustifolia subsp. angustifolia%
L. angustifolia subsp. pyrenaica
L. angustifolia subsp. delphinensis
L. lanata%
L. latifolia%
Section Pterostoechas
## $
## $
## $
## $
## $
#
#
#
#
#
$
$
$
$
$
$
$
$
$
$
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
$
$
$
$
$
$
$
$
$
$
!
!
!
!
!
!
!
!
!
!
L. canariensis%
$
!
!
!
!
!
!
!
## #
!
#
$
#
!
!
!
!
L.
L.
L.
L.
L.
L.
$
$
$
!
$
$
!
!
$
!
!
!
!
!
!
!
!
!
!
!
$
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
## ## !
## ## $
! ! !
## ! !
## ! !
## ## #
## ## !
## ## #
! # #
# $ #
# ## $
## ## #
!
!
!
#
!
!
!
!
!
!
!
!
!
!
#
#
!
!
!
!
!
$
!
!
Compound
Texon
Section Stoechas
L. stoechas subsp. stoechas%
L. stoechas subsp. luiseiri
L. viridis%
Section Dentata
coronopifolia
maroccana%
mairei var. mairei%
minutolii var. minutolii%
multixda
rotundifolia
1/2
3
4/5
6
1001
2continued
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
Flavone aglycones
8-OH
Flavone 7- (external)
glycosides
Fla.
6-OH
C-gly.
Flavone
glycosides
Flavone glycosides
1002
Compound
Texon
Flavone aglycones
8-OH
Flavone 7- (external)
glycosides
Fla. 8-OH Flavone
6-OH
C-gly. 8- glycosides
Flavone
glycosides
Flavone glycosides
1/2
3
4/5
6
7/8
9
10
11
12
Section Subnuda
L. aristibracteata%
L. dhofarensis%
L. subnuda
!
$
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
Section Chaetostachys
L. bipinnata%
$
!
!
!
!
!
!
Sabaudia group
S. atriplicifolia
$
!
$
#
!
!
!
13
17
18
19
20
21
# ## # $ $
## # ## ! $
## # # ## $
$
!
$
!
!
!
!
!
!
!
!
!
#
!
!
!
## ## #
$
$
$
!
!
!
$
!
!
!
#
## !
!
!
!
!
14
!
15
16
%Extract subjected to electrophoresis; Fla. C-Gly.: Flavone C-glycoside; !Flavonoid not detected by HPLC; $, #, ## Relative amounts of #avonoids
present (low- medium- or high-UV absorbance of the #avonoid peak, respectively).
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
Table 3*continued
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
1003
Table 4
The distribution of #avonoids in additional species, subspecies and varieties of Lavandula and Sabaudia
based on PC results only
Taxon
Flavone glycosides
8-OH Flavone glycosides
1
2
4
5
12
16
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
!
!
!
!
!
!
!
!
!
!
Section Dentata
L. dentata var. candicans
#
#
#
#
!
!
Section Pterostoechas
L. antineae
L. buchii var. buchii
L. buchii var. gracile
L. mairei var. antiatlantica
L. minutolii var. tenuipinna
L. pinnata
L. tenuisecta
$
$
$
$
!
$
$
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
#
#
#
!
#
#
#
#
#
#
#
#
#
#
Section Subnudae
L. nimmoi
$
!
!
!
#
#
Sabaudia group
S. erythreae
#
#
!
!
!
#
Section Stoechas
L. stoechas subsp.
L. stoechas subsp.
L. stoechas subsp.
L. stoechas subsp.
L. stoechas subsp.
atlantica
cariensis
lusitanica
pedunculata
sampaiana
was restricted to certain sections. For example, the #avone diglycosides, luteolin
7,4@-diglucuronide (7) and 7-glucoside-4@-glucuronide (8) were found only in section
Lavandula and the #avone C-glycoside, vitexin (11), only in sections Stoechas and
Dentata. 8-Hydroxy#avone glycosides occurred widely in sections Pterostoechas,
Subnuda and Chaetostachys and in Sabaudia, whereas 6-hydroxy#avone glycosides
were restricted to section Dentata. As to leaf surface #avonoids, apigenin (18) and
genkwanin (19) were regularly detected in members of sections Stoechas, Dentata and
Lavandula, whereas the highly methoxylated external #avones 20 and 21 were found in
sections Stoechas, Pterostoechas, Subnudae and Chaetostachys.
4. Discussion
The combined use of 2-D paper chromatography and HPLC analysis has provided
both a detailed and comprehensive survey of the leaf #avonoids in Lavandula. The
1004
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
results show a strong correlation between the accumulation of certain #avonoid
classes and groupings of species, which is congruent with the present sectional
classi"cation. The variation found at the lower taxonomic ranks was either not great
enough to be informative or appeared to be random and not correlated to any
taxonomic entity presently recognised. However, #avonoid patterns do appear to be
of systematic use at the sectional level and above in Lavandula. The distribution of the
di!erent #avonoid classes in the sections of Lavandula and in Sabaudia is presented in
Table 5. In this Table accumulation rather than presence of a #avonoid class was often
taken as a character, because chemotaxonomically accumulation of a chemical feature
is more important than mere presence. The sensitivity of HPLC can reveal presence of
trace compounds which serve as precursors of the ones that accumulate. For instance,
small amounts of a #avone 7-monoglycoside were present in section Pterostoechas,
Subnuda and Chaetostachys, but only 8-hydroxylated #avone glycosides were accumulated in these taxa.
Three major groupings of taxa can be identi"ed using #avonoid features as
characters. The "rst group corresponds to sections Stoechas, Dentata and Lavandula,
which are characterised by the accumulation of #avone 7-monoglycosides. However,
each of these sections can be distinguished by its overall #avonoid pro"le (see Table 5).
Thus, Lavandula is unique in producing #avonoid diglycosides, whereas sections
Dentata and Stoechas both contain #avone C-glycosides. But section Dentata di!ers
from section Stoechas in the presence of 6-hydroxylated #avone 7-glycosides. The
second chemical group within the genus contains sections Pterostoechas, Subnuda and
Chaetostachys, which are characterised by an accumulation of 8-hydroxylated #avone
7-and 8-glycosides. There are small individual di!erences among the #avonoid pro"les of the species in these sections, but no overall distinction in #avonoid patterns
among the three sections.
The third group corresponds to those taxa assigned to the genus Sabaudia, which
accumulate both 8-hydroxylated #avone 7-glycosides and #avone 7-glycosides. It is
therefore characterised by the presence of compounds which otherwise are accumulated only in group one or group two. Flavone 7-glycosides are the most common type
of #avonoid in the Lamiaceae, and therefore are not very informative about relationships within this family (TomaH s-BarberaH n et al., 1988). 8-Hydroxylated #avone glycosides, on the other hand, are uncommon in the family. Indeed, in subfamily
Nepetoideae they have only been found in the genus Lavandula (El Garf et al., 1999),
so that the presence of this class of #avonoid in Sabaudia is a very strong indication
that this taxon is very closely related to Lavandula, at least to those sections that
contain 8-hydroxy#avone glycosides. It is possible that Sabaudia is basal to all
sections of Lavandula, but that the species of sections Stoechas, Dentata and Lavandula
have all without exception lost the ability to synthesise the 8-hydroxy#avones.
However, this does not seem very likely, as the ability to produce these #avonoids has
not been lost in any of the taxa belonging to the other three sections. It seems more
probable that a predecessor of Sabaudia has evolved the ability to produce 8hydroxy#avone 7-glycosides and that this taxon is also basal to sections Pterostoechas, Subnuda and Chaetostachys, but not to the other three sections of Lavandula.
If this is the case, the immediate predecessor of Pterostoechas, Subnuda and Chaetos-
Taxon
Accumulation of
#avone 7-Omonoglycosides
(Compounds 1}6)
Presence of
#avone di-Oglycosides
(Compounds 7
and 8)
Accumulation of
6-hydroxylated
#avone 7-Oglycosides (Compounds 9 and 10)
Presence of
#avone C-glycosides (Compound
11)
Accumulation
of 8-hydroxylated
#avone 8-Oglycosides
(Compounds
12}15)
Accumulation of
8-hydroxylated
#avone 7-Oglycosides
(Compounds
16 and 17)
Presence of
external #avones
(Compounds
18}21)
Section Stoechas
Section Dentata
Section Lavandula
Section Pterostoechas
Section Subnudae
Section Chaetostachys
Sabaudia group
#
#
#
!
!
!
#
!
!
#
!
!
!
!
!
#
!
!
!
!
!
#
#
!
!
!
!
!
!
!
!
#
#
#
!
!
!
!
#
#
#
#
#
#
#
#
#
#
?
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
Table 5
The distribution of di!erent #avonoid classes in sections of Lavandula and in Sabaudia
1005
1006
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
tachys must also have evolved separately the ability to produce 8-hydroxy#avones
8-glycosides.
The major groupings of sections suggested by the #avonoid data are highly
congruent with the major groupings suggested by other data sets such as gross
morphology, carpology, molecular sequence data and palynology (Upson, 1997). At
present, this grouping of sections is not re#ected in any classi"cations of the genus, but
evidently these data need to be incorporated. The results obtained here provide
further compelling evidence to support the recognition of these groupings. The rank at
which to recognise these groups is a more complex question and in the taxonomic
hierarchy they probably represent either subgenera or even distinct generic groupings.
Acknowledgements
The University of Reading is gratefully acknowledged for funding to T.M.U.
through a postgraduate scholarship. We thank the following people for their help in
supplying plant material for use in this study: J. Head (National Plant Collection
holder of Lavandula under the National Council for the Conservation of Plants and
Gardens Scheme), W. Lobin (Botanischer Garten der UniversitaK t Bonn), A.G. Miller
(Royal Botanic Garden, Edinburgh), H.D.V. Prendergast (Royal Botanic Gardens,
Kew), N.J. Singh (Botanical Survey of India) and the Curators of the following
herbaria: BM, C, E, M and O. Thanks to Dr. Stephen Jury of the University of
Reading for help and advice in undertaking this work.
References
Boelens, M.H., 1995. Chemical and sensory evaluation of Lavandula oils. Perfumer & Flavorist 20, 23}51.
Brummit, R.K., Powell, C.E., 1992. Authors of Plant Names. Royal Botanic Gardens, Kew, UK.
Cantino, P.D., Harley, R.M., Wagsta!, S.J., 1992. Genera of Labiatae: status and classi"cation. In: Harley,
R.M., Reynolds, T. (Eds.), Advances in Labiate Science. Royal Botanic Gardens, Kew, UK, pp. 511}522.
Chaytor, D.A., 1937. A taxonomic study of the genus Lavandula. J. Linnaean Soc. Botany 51, 153}204.
Cufodontis, G., 1962. Enumeratio plantarum Aethiopiae spermatophyta. Bulletin du Jardin Botanique de
l'ED tat. Bulletin van den Rijksplantentuin. Brussels, 32 (Suppl.) pp. 806}807.
El-Garf, I., Grayer, R.J., Kite, G.C., Veitch, N.C., 1999. Hypolaetin 8-O-glucuronide and related #avonoids
from Lavandula coronopifolia and L. pubescens. Biochem. Syst. Ecology 27, 843}846.
Erdtman, G., 1945. Pollen morphology and plant taxonomy IV. Labiatae, Verbenaceae and Avicenniaceae.
Svensk Botanisk Tidskrift 39, 279}285.
Ferreres, F., BarberaH n, F.A.T., TomaH s, F., 1986. Flavonoids from Lavandula dentata. Fitoterapia 57,
199}200.
Grayer, R.J., Kite, G.C., Goldstone, F.J., Bryan, S.E., Paton, A., Putievsky, E., 1996. Infraspeci"c taxonomy
and essential oil chemotypes in sweet basil. Ocimum basilicum. Phytochemistry 43, 1033}1039.
Harborne, J.B., 1998. Phytochemical Methods, 3rd Edition, Chapman & Hall, London.
Hegnauer, R., 1989. Chemotaxonomie der P#anzen, Vol. 8. BirkhaK user, Basel.
Kaufmann, M., Wink, M., 1994. Molecular systematics of the Nepetoideae (Labiatae): phylogenetic
implications from rbcL gene sequences. Zeitschrift fuK r Naturforschung C49, 635}645.
Mabry, T.J., Markham, K.R., Thomas, M.B., 1970. The Systematic Identi"cation of Flavonoids. Springer,
New York.
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Miller, A.G., 1985. The genus Lavandula in Arabia and Tropical N.E. Africa. Notes of the Royal Botanic
Garden. Edinburgh 42, 503}528.
Ross, J.D., Sombrero, C., 1991. Environmental control of essential oil production in Mediterranean plants.
In: Harborne, J.B., TomaH s-BarberaH n, F.A. (Eds.), Ecological Chemistry and Biochemistry of Plant
Terpenoids. Clarendon Press, Oxford, pp. 83}94.
TomaH s-BarberaH n, F.A., Grayer-Barkmeijer, R.J., Gil, M.I., Harborne, J.B., 1988. Distribution of 6-hydroxy-,
6-methoxy- and 8-hydroxy#avone glycosides in the Labiatae, the Scrophulariaceae and related families.
Phytochemistry 27, 2631}2645.
Upson, T.M., 1997. Systematics of the genus Lavandula L. (Lamiaceae). Ph.D. Thesis, University of Reading.
Wagsta!, S.J., Olmstead, R.G., Cantino, P.D., 1995. Parsimony analysis of chloroplast DNA variation in
subfamily Nepetoideae. Am. J. Botany 82, 886}892.
Xaver, H., Andary, C., 1988. Polyphenols of Lavandula stoechas L. Bulletin Liaison Group Polyphenols 133,
624}626.
Leaf #avonoids as systematic characters
in the genera Lavandula and Sabaudia
Tim M. Upson!,1, ReneH e J. Grayer", Jennifer R. Greenham!,
Christine A. Williams!,*, Farag Al-Ghamdi!, Fen-Hui Chen!
!Department of Botany, University of Reading, Whiteknights, Reading RG6 6AS, UK
"Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK
Received 28 September 1999; accepted 3 December 1999
Abstract
A comprehensive survey of the leaf #avonoids of the genus Lavandula and the related
Sabaudia group was carried out using two-dimensional paper chromatography and highperformance liquid chromatography. The #avonoid patterns obtained were found to be
systematically informative at the infrageneric level. Three main groupings were identi"ed: the
"rst containing sections Lavandula, Dentata and Stoechas characterised by the accumulation of
#avone 7-glycosides; the second containing sections Pterostoechas, Subnuda and Chaetostachys
characterised by the accumulation of 8-hydroxylated #avone 7- and 8-glycosides; the third
encompassing the Sabaudia group and accumulating both #avone and 8-hydroxylated #avone
7-glycosides. Such a grouping of taxa is congruent with data from other disciplines, although it
is not recognised in any present classi"cations. The taxonomic and evolutionary implications of
the #avonoid data are discussed. ( 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Lavandula; Sabaudia; Lavender; Lamiaceae; Flavonoids; Chemotaxonomy
1. Introduction
The genus Lavandula (lavender, Lamiaceae) is distributed from the Canary and
Cape Verde Islands and Madeira, across the Mediterranean Basin, North Africa,
* Corresponding author. Tel.: #44-118-9-3181-68; fax: #44-118-9-7536-76.
E-mail address: christine.williams@reading.ac.uk (C.A. Williams)
1 Present address: Cambridge University Botanic Garden, Cory Lodge, Bateman Street, Cambridge CB2
1JF, UK.
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 1 3 - 2
992
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
South}West Asia, the Arabian Peninsula and tropical NE Africa with a disjunction to
India. Although the name of the genus is familiar to many people, only a few species
are well known, e.g. L. angustifolia Miller or English lavender, L. latifolia or spike
lavender, and L. stoechas or French lavender. The English lavender is widely cultivated in gardens and is also an important essential oil crop from which many cultivars
have been selected.
The last revision covering the whole genus was undertaken more than 60 years ago
by Chaytor (1937), but more recently there have been a number of #oristic revisions
covering certain geographical areas, e.g. by Miller (1985) on the Arabian and NE
tropical African species. Based on Chaytor's work and that of others, six sections can
be recognized in Lavandula: Stoechas Ging. (3 spp.), Lavandula (3 spp. ), Dentata (1 sp.),
Pterostoechas (15 spp.), Subnuda (8 spp.) and Chaetostachys (2 spp.).
On the basis of its hexacolpate pollen (Erdtman, 1945) and accumulation of
essential oils (Hegnauer, 1989), Lavandula clearly belongs to the subfamily
Nepetoideae of the Lamiaceae. According to phylogenetic and molecular studies,
Lavandula is a distinctive clade in the Nepetoideae without close relatives (Cantino
et al., 1992; Kaufmann and Wink, 1994; Wagsta! et al., 1995). The nearest relative
appears to be the tribe Ocimeae. However, there is a small genus of two or three
species native to the Arabian Peninsula and NE tropical Africa, called Sabaudia,
which has sometimes been treated as part of Lavandula (Cufodontis, 1962), although
its tribal position within the Lamiaceae has not yet been fully established.
A vast literature exists on the essential oils in Lavandula, reviewed by Boelens
(1995). For the taxonomist the use of essential oils as characters for the classi"cation is
limited by inherent problems of natural variability, although at lower taxonomic
levels this can be used to help recognize cultivars (Grayer et al., 1996). The chemical
composition and ratios of the individual components making up the oils are also
known to change in response to environmental conditions, such as water and nutrient
stress or time of year (Ross and Sombrero, 1991). These inherent problems of variation
mean that other classes of chemical constituents such as #avonoids are often of greater
use and signi"cance to the systematist.
The utility of #avonoids within the Lamiaceae has been demonstrated at various
taxonomic levels, including the assessment of generic relationships within subfamilies,
generic a$nities of species, and even in verifying the parents of hybrids (reviewed by
TomaH s-BarberaH n and Gil, 1992). With regard to the genus Lavandula, no #avonoid
survey of the genus has yet been undertaken, although some species have been
investigated as part of wider taxonomic surveys of the family. The following
#avonoids were reported from L. dentata (Ferreres et al., 1986): genkwanin (apigenin
7-methyl ether), luteolin, apigenin, luteolin 7-glucoside, apigenin 7-glucoside, luteolin
7-rutinoside, vitexin and vicenin-2. Several species of Lavandula were investigated as
part of a survey on the occurrence of 6-hydroxy-, 6-methoxy- and 8-hydroxy#avone
glycosides within the Lamiaceae and related families (TomaH s-BarberaH n et al., 1988),
but no total #avonoid pro"les were given. The major #avonoids in L. stoechas were
apigenin 7-glucoside, luteolin, luteolin 7-glucoside and luteolin 7-glucuronide (Xaver
and Andary, 1988). Very recently, a number of #avone glycosides with extra hydroxylation in the 8-position were reported from L. pubescens and L. coronopifolia,
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
993
species both belonging to section Pterostoechas. These compounds were the 7glucoside and 8-glucuronide of hypolaetin, and the 8-glucuronides of hypolaetin
4@-methyl ether and isoscutellarein (El Garf et al., 1999). Glycosides of 8-hydroxyflavones are uncommon plant constituents, especially the 8-glycosides, and in several
cases have provided useful characters at the sectional level within genera of the
Lamiaceae (TomaH s-BarberaH n et al., 1988; TomaH s-BarberaH n and Gil, 1992). To see
whether that is also the case in the genus Lavandula, we have surveyed a large
proportion of the species of the genus for the presence or absence of these and other
#avonoids, and also studied representatives of Sabaudia in the hope that #avonoid
pro"les would help in clarifying relationships between this genus and Lavandula.
2. Materials and methods
2.1. Plant material
Specimens and voucher details for the taxa surveyed are presented in Table 1. The
fresh material used in the investigations was "rst grown in a greenhouse and then
moved outside to grow under natural daylight for six weeks before extraction.
2.2. Extraction
A few chopped leaves of each sample were placed in a test tube, covered with, ca.
5 ml of 100% MeOH for fresh material or 70% aqueous MeOH for dry herbarium
material, and placed in a heating block at 703C for about 5 min. This procedure kills
the plant tissue preventing enzymic oxidation or hydrolysis. The samples were left to
extract for at least 8 h or overnight. Excess chlorophyll was removed using petroleum
ether (BP 40}603) before the extracts were evaporated to dryness on watchglasses.
2.3. Two-dimensional (2-D) -paper chromatography
The dried extracts were reconstituted using 80% MeOH and run two-dimensionally on quarter sheets of Whatman no. 1 chromatography paper. The chromatograms were run by descent in BAW (n-BuOH, HOAc, H O"4 : 1 : 5; v/v; upper
2
layer) "rst direction, and in 15% aqueous HOAc or H O in the second direction.
2
After drying, the chromatograms were viewed in UV light (366 nm) "rst without and
later in the presence of ammonia vapour. The colours of the spots before and after
NH were recorded and R -values calculated.
3
&
2.4. Paper electrophoresis
Extracts were applied as spots along a central axis on a sheet of Whatman no.
3 chromatography paper, measuring 19]28.5 cm. A spot of rutin (quercetin 3rutinoside) was used as a negative control, and quercetin 3-glucuronide as a positive
Table 1
Specimen and voucher details for the taxa surveyed. Accession numbers are given for those living specimens held in the collection at the University of Reading;
herbarium acronyms refer to vouchers and follow Holmgren, Keuken and Scho"eld (1981). Authority abbreviations follow Brummitt and Powell (1992).
L"living material used, H"herbarium material used
994
Taxa
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
Specimen and voucher details
Section Lavandula
L. angustifolia Mill. subsp. angustifolia
L
L. angustifolia Mill. subsp. pyrenaica (DC.) Masclans
L
L. angustifolia Mill. subsp. delphinensis (Jord.)
O. Bolòs & Vigo
L. latifolia Medic.
L
L. lanata Boiss.
L
Section Stoechas
L. stoechas L. subsp. stoechas
L
L. stoechas L. subsp. atlantica Braun-Blanq
L
L. stoechas L. subsp. cariensis (Boiss.) Rozeira
L. stoechas L. subsp. luisieri (Rozeira) Rozeira
L
L
L
Cultivated ex. Kiev Botanic Garden, Russia (RNG); Cultivated ex. Tsukuba Medicinal
Plant Research Station, Japan (RNG); Cultivated variant (RNG)
Cultivated ex. Botanischer Garten Kiel (RNG); Spain, Prov. of Huesca, Pyrenaen PlanBenasque ex. Botanischer Garten Berlin Dahlem (RNG); France, Frankreich Pyrenees
Orientalis ex. Botanischer Garten Berlin Dahlem (RNG)
Cultivated ex. Bundesgarten Wien } Alpengarten Im Belvedere (RNG)
France, Prov. of Aude, Montagne de Tauch ex. Jardin Botanique Leige (RNG); France,
Aude Fore( t de Rives-Hautes ex. Leige (RNG); Spain, Prov. Gerona Angles, ex. Botanischer Garten Berlin Dahlem (RNG); Italy, Siena, Pomona in Chianti ex. Jardin Botanico
Siena (RNG); France Angers ex. Instituto Botanico Siena (RNG)
Spain, Prov. of Granada, Sierra Nevada ex. Botanischer Garten Berlin Dahlem (RNG);
Cultivated ex. Jardin Botanique Ronen (RNG); Spain, Prov. of Malaga, S.L. Jury 13138
(RNG)
Corsica, Mt. Tomboni, L. Springate 93.289 (RNG) Crete, J.D. Ross s.n. (RNG); Turkey,
Mugla province, SW of Marmaris, L.C. Jury 334 (RNG); France, Angers ex. Jardin
Botanique de Dijon (RNG); Spain, Prov. of Granada, Sierra Nevada ex. Jardin Botanico
Cordoba (RNG); Morocco, SE of Tetouan, 1 km N of Oued Laou, S.L. Jury 12408 et al.
(RNG)
Morocco, Rif Central, 17 km S. Ketama, S.L. Jury 11566 et al. (RNG); Morocco, Haut
Ouerrha, 47 km S. of Ketama, S.L. Jury 11575 et al. (RNG)
NW Turkey, Balikesir, Haziren s.n. (RNG)
Portugal ex. Jardim Botanico Porto (RNG); Portugal, Algarve, Serra do Caldeira8 o ex.
Jardim Botanico Lisboa (RNG) Spain, Sa de CoH rdoba ex. Jardin Botanico Cordoba (RNG);
Portugal, Coimbra ex. Jardim Botanico Coimbra (RNG)
L
L. stoechas L. subsp. pedunculata (Mill.) Samp.
ex. Rozeira
L. stoechas L. subsp. sampaiana Rozeira
L
L
L. viridis L@HeH r.
L
Section Dentata
L. dentata L. var. dentata
L
L. dentata L. var. candicans Batt.
L
Section Pterostoechas
L. antineae Maire
H
L. buchii Webb & Berthel. var. buchii
H
L. buchii Webb & Berthel. var. gracile LeoH n
L
L. canariensis Mill.
L
L. coronopifolia Poir.
H
L
L. mairei Humbert var. mairei
L
L. mairei Humbert var. antiatlantica Maire
H
L
Portugal, Prov. Baixo Alentejo, Reguengos de Monsaraz ex. Jardim Botanico de Lisboa
(RNG)
Spain, Prov. of Leòn, La Magdalena ex. Jardin Botanico Bilbao (RNG); Spain, Prov. of
Avila, Sierra de Gredos ex. Botanisher Garten OsnabruK ck (RNG)
Portugal, Prov. Alto Alentejo, Marva8 o ex. Jardim Botanico Lisboa (RNG) Portugal, nr.
Coimbra ex. Jardim Botanico Coimbra (RNG)
Portugal, outside Barrada ex Joan Head 91.10 (RNG); Portugal, Barranco Velho ex. Joan
Head 91.08 (RNG); Cultivated ex Jardin Botanico Nice (RNG)
Balearic Islands, Mallorca (RNG); Morocco, S. of Tetouan on rd. to Oued Laou, Cap
Mazari, S.L. Jury 12383 et al. (RNG); Morocco, N. of Agadir, rd. to Immouzzer, S.L. Jury
11984 et al. (RNG); Cultivated ex. Jardin Botanico Barcelona (RNG)
Morocco, NW of Agadir, Cap Rhir, S.L. Jury 12000 et al. (RNG); Morocco, nr. Al
Hoceima, S.L. Jury 13533 (RNG)
Algeria, Hoggar Mts, Miller 4144 et al. (E); Algeria, Hoggar Massif, Oued Ideles, D.
Podlech 36932 (M)
Canary Islands, Tenerife: Anaga Peninsula, near Las Carboneras, T.M. Upson 299 (RNG);
Anaga Peninsula, NW of Taganana, Playa de Benijo, T.M. Upson 307 (RNG)
Canary Islands, Tenerife, Teno Region ex. Joan Head (RNG); Teno Region, c. 6 km SW of
Tamaimo, T.M. Upson 301 (RNG); Teno Region, Mirador de Don Pompeyo, nr.
Buenavista, T.M. Upson 297 (RNG)
Cultivated ex. Norfolk Lavender (RNG); Canary Islands, Gran Canaria, Arucas, Joan
Head 93.21 (RNG)
Canary Islands, Tenerife, above San Juan, T.M. Upson 298 (RNG)
Morocco: SW Anti-Atlas, c.27 W of Tata, S.L. Jury 14464 et al. (RNG); SW Anti-Atlas, c.
20 km W of Tata, S.L. Jury 14448 et al. (RNG)
Morocco: 24 km N. of Er Rachidia, S.L. Jury 14612 (RNG); c. 26 km ESE of Tinerhir, S.L.
Jury 14589 (RNG)
Morocco, Gorges du Ziz, S.L. Jury 14614 (RNG)
Morocco, SW Anti-Atlas: W of Tata, S.L. Jury 14463a et al. (RNG); 27 km NE of
Tafraoute, S.L. Jury 14384 et al. (RNG); SE of AmK t-Baha, S.L. Jury 14385 et al. (RNG)
995
2continued
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
L. stoechas L. subsp. lusitanica (Chaytor) Rozeira
996
Table*continued
Taxa
Specimen and voucher details
L
L. minutolii Bolle var. minutolii
L
L. minutolii Bolle var. tenuipinna Svent.
L. multixda L.
L. pinnata L. ff.
H
L
Morocco: Gorges du Ziz, S.L. Jury 14615 (RNG); Morocco: Immouzzer Valley, 10 km
N of Agadir, S.L. Jury 14245 (RNG); Morocco: High Atlas, c. 12 km NNE of Ijoukak, S.L.
Jury 14200 (RNG)
Canary Islands, Gran Canaria, Joan Head s.n. (RNG); Canary Islands, Gran Canaria,
Roque Bentriga ex. Botanischer Garten Berlin Dahlem (RNG)
Canary Islands, Tenerife, Teno Region, near Masca, T.M. Upson 295 (RNG)
Culivated ex. Jardin Botanique De Dijon, France (RNG)
Cultivated ex. Orto Botanico Torino (RNG); Cultivated ex. Botanischer Garten Innsbruck
(RNG)
Cape Verde Island ex. Botanischer Garten Bonn (RNG)
Cape Verde Islands, mountains SW of the village Ribeira Grande, below Faja8 Bedonda,
26.1.1982, C. Brochmann 533/82 (O)
High Atlas, Toubkal range, side of gorge from refuge Neltner to Imlil village, 14.7.1966,
T.K. Thorp no. 95 (BM)
L. rotundifolia Benth.
L
H
L. tenuisecta Coss. ex. Ball
H
Section Subnuda
L. aristibracteata A.G. Mill.
L. dhofarensis A.G. Mill. subsp. dhofarensis
L. nimmoi Benth.
L
L
H
L. subnuda Benth.
L
Section Chaetostachys
L. bipinnata Kuntze
L
India, Prov. of Maharashtra, Poona District, Vede-Bowdhan ex. Botanical Survey of India
(BSI, RNG)
Sabaudia group
S. atriplicifolia (Benth.) Chiov.
S. erythreae Chiov.
H
H
Yemen Arab Republic, Kaukaban, J.R.I. Wood Y/75/311 (BM)
Ethiopia (Eritrea), Monti Lesa, A. Pappi (C)
Somalia, Surundi Hills, M. Thulin 9051 (RNG)
Oman, Dhofar, Wadi Sahalnawt, nr. Salalah, A.G. Miller 6158 (RNG)
Socotra, base of escarpment c. 6 km SE of Qalansiyah, 23.2.1989, A.G. Miller et al. 8395
(E)
N. Oman, Wadi Mistral, S. Gbazdrfai 2482 (RNG); Oman, Musandam by road to Khawr
Niad, H.D.V. Prendergast (RNG)
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
L. maroccana Murb.
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
997
control. The paper was subjected to electrophoresis at pH 4.4, using a sodium
acetate/acetic acid bu!er at 400 V for about 2 h.
2.5. Preparative paper chromatgraphy (PPC)
Flavonoids were separated from rosmarinic acid and other ca!eic acid derivatives
which interfered with the HPLC analysis of crude extracts by means of PPC. Each
crude extract was applied as a streak onto one quarter sheet of Whatman no.
3 chromatography paper. This was run by descent in 15% HOAc. The bottom half of
the paper which showed dark #avonoid bands in UV light (roughly from the origin to,
ca. R 0.50) was cut out, cut into 2 cm squares, and these were eluted in 80% MeOH
&
for 3}4 h. The top half of the paper (R -value above 0.50) which showed #uorescent
&
blue bands of ca!eic acid derivatives such as rosmarinic acid (R 0.58 in 15% HOAc)
&
was not eluted. The #avonoid eluate was evaporated and resuspended in ca. 0.8 ml of
80% MeOH. This was "ltered using a 13 mm Gelman Acrodisc, pore size 5 lm, ready
for HPLC analysis. A few extracts were further separated by PPC to isolate
#avonoids, using the solvent BAW (see above) as a second puri"cation step.
2.6. HPLC
The HPLC system consisted of a Waters 600E systems controller with a Waters 996
photodiode array detector. Injection of the extracts (40 ll) took place with a Waters
717 autosampler. Millennium software was used to record and analyse the results.
Two di!erent elution programmes were used; a LiChrospher 100RP-18 column was
employed for programme 1 and a Bondapak Phenyl RP-18 column for programme 2.
Both columns measured 4.0 mm (i.d.) ]250 mm with a 5 lm particle size. Elution
took place by means of two solvents, solvent A consisting of 2% of aqueous HOAc
and solvent B of MeOH, HOAc, H O, 18: 1: 1, and elution started with 25% of B at
2
t"0. For programme 1 there was a linear gradient reaching 100% B at t"20 min,
but for programme 2 it reached 65% B at t"23 min. In both programmes this was
followed by an isocratic elution with 100% B for 5 or 1 min, respectively, before going
back to the initial conditions. The #ow rate was 1.0 ml/min, and the temperature of
the column was 253C.
2.7. Identixcation of yavonoids
The #avonoids were identi"ed by comparing R -values on paper chromatograms,
&
colours of spots, HPLC R s and UV spectra of the compounds in the extracts with
5
those of standard markers. Puri"ed compounds were subjected to UV spectroscopy
using shift reagents to determine substitution patterns (Mabry et al., 1970), and to acid
hydrolysis to determine the aglycone and sugar moieties (Harborne, 1998). The
following #avonoid standards were available for comparison: luteolin 7-glucoside,
apigenin 7-glucoside, vitexin, apigenin, genkwanin (all purchased from Apin); luteolin
7-glucuronide, apigenin 7-glucuronide, (#avonoid collection at Reading University);
hypolaetin 8-glucuronide, its 4@-methyl ether, isoscutellarein 8-glucuronide,
998
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
hypolaetin 7-glucoside (#avonoid collection at Kew); xanthomicrol and salvigenin
(kindly provided to R.J.G. by Prof. E. Wollenweber).
3. Results
The extracts of 23 species of Lavandula and two of Sabaudia were examined by
means of 2-D paper chromatography. In addition, seven di!erent subspecies of L.
stoechas were studied together with three subspecies of L. angustifolia and two
varieties of L. dentata, L. buchii, L. mairei and L. minutolii. Besides, for many of the taxa
more than one accession was examined (see Table 1). Extracts of 13 species, representing all sections, were subjected to paper electrophoresis to check for the presence of
#avonoid glucuronides which are characteristic constituents of the family Lamiaceae
(TomaH s BarberaH n et al., 1988). The crude extracts used for the 2-D paper chromatograms were also analysed by HPLC. However, the HPLC traces did not give clear
results, because of the presence of large amounts of rosmarinic acid and related
substances in the extracts, which masked many of the #avonoid peaks and deformed
their UV spectra. For this reason about half of the extracts, representing all six
sections of Lavandula and all species of Sabaudia, were puri"ed by means of PPC to
separate the #avonoids from rosmarinic acid. HPLC of these puri"ed #avonoid
fractions gave much better results.
To identify some of the compounds, an extract of L. angustifolia ssp. pyrenaica was
used to isolate #avonoids by means of preparative paper chromatography and
preparative paper electrophoresis. Ten #avonoids were obtained, 1+8, 18 and 19.
Their colours on the chromatograms before and after fuming with ammonia, R &
values in two or three solvents, HPLC retention times in two di!erent solvent systems
and diode array UV spectra were determined (see Table 2). In addition, 1, 2, 4, 5, 7 and
8 were studied by UV spectroscopy using shift reagents (Mabry et al., 1970) and by
means of acid hydrolysis of the compounds and subsequent analysis of the aglycones
and sugars (Harborne, 1998). In this way, 1 and 2 were identi"ed as the 7-glucoside
and 7-glucuronide of luteolin, respectively, and 4 and 5 as apigenin 7-glucoside and
7-glucuronide. These identi"cations were con"rmed by means of co-chromatography
with standard markers on paper and by HPLC. After partial and total hydrolysis of
7 and 8 and analysis of the hydrolysis products, these compounds were tentatively
identi"ed as luteolin 7,4@-di-glucuronide and luteolin 7-glucoside-4@-glucuronide. No
standards were available for comparison. Not enough pure compound for 3 and 6 was
obtained for complete identi"cation. HPLC of these compounds revealed that 3 had
a similar UV spectrum to 1 and 2, but a longer retention time. The colour of 3 on
chromatograms in UV light after fuming with ammonia (yellow}green) and the UV
spectrum suggested that it may be a chrysoeriol 7-glycoside. In the same way, 6 may
be a glycoside of a methylated apigenin derivative. Compounds 18 and 19 were
identi"ed by means of co-chromatography with standards as the #avone aglycones,
apigenin and genkwanin.Table 2 shows another 11 #avonoids, 9}17, 20 and 21,
frequently encountered in extracts of Lavandula species, which were completely or
partly identi"ed by means of co-chromatography with standards using HPLC and
Table 2
Chromatographic and UV spectral data of #avonoids from Lavandula and Sabaudia species!
Compound
9
10
11
12
13
14
15
16
17
18
19
20
21
Colour
in UV
Luteolin 7-O-glucoside
D
Luteolin 7-O-glucuronide D
Chrysoeriol 7-O-glycosideH D
Apigenin 7-O-glucoside
D
Apigenin 7-O-glucuronide D
Derivative of 4 or 5
n.d.
Luteolin 7,4@-di-O-glucur.H D
Luteolin 7-O-glucosideD
4@-O-glucuronideH
6-OH-luteolin 7-OD
glycosideH
Scutellarein 7-O-glycosideH D
Vitexin
D
Hypolaetin 8-O-gluD
curonide
Derivative of 12
D
Hypolaetin 4@-methyl
D
ether 8-O-glucuronide
Isoscutellarein 8-O-glucur. D
Hypolaetin 7-O-glucoside D
Isoscutellarein 7-OD
glycoside
Apigenin
D
Genkwanin
D
Xanthomicrol
D
Salvigenin
D
R in BAW
&
R in 15%
&
HOAc
R in H O
&
2
R in solv.
5
1 (min)
R in solv.2
5
(min)
UV spectrum
(j
nm)
.!9
Y
Y
GnY
dYGn
dYGn
n.d.
Br
Br
0.40
0.32
0.42
0.58
0.50
n.d.
0.15
0.15
0.12
0.15
n.d
0.25
0.28
n.d.
0.40
0.40
0.02
0.09
n.d.
0.04
0.15
n.d.
0.69
0.69
14.2
14.1
15.9
15.7
15.7
17.2
n.d.
n.d.
17.4
17.4
21.0
20.3
20.3
23.7
12.8
12.8
256,
256,
252,
268,
268,
268,
269,
269,
D
0.25
0.08
n.d.
12.6
n.d.
285, 348
D
dY
D
0.35
0.40
0.40
0.13
0.30
0.12
n.d.
n.d
n.d.
14.1
13.2
15.9
n.d.
n.d.
n.d.
285, 338
269, 338
256sh, 271, 354
D
D
0.45
0.48
0.25
0.14
n.d.
n.d.
16.7
17.3
n.d.
n.d.
256sh, 271, 354
257, 271, 352
D
D
D
0.55
0.20
0.35
0.20
0.08
0.15
n.d.
n.d.
n.d.
17.4
13.9
15.4
n.d.
n.d.
n.d.
271, 343
276, 300sh, 343
276, 306, 328sh
Ygn
GnY
D
D
0.85
0.90
0.90
0.90
0.08
0.08
0.15
0.15
n.d.
n.d.
n.d.
n.d.
20.5
24.2
23.0
24.9
26.8
32.5
n.d.
n.d.
Colour
UV#NH
3
268,
268,
281,
276,
267sh, 350
267sh, 350
267sh, 348
338
338
338
336
336
338
338
290sh, 333
333
999
!Solv. 1 and Solv. 2: HPLC solvent programs 1 and 2, respectively. For composition of the solvents used for HPLC and PC, see Experimental.
Abbreviations: D"dark; Y"yellow; L"light; Gn"green; d"dull; Br"brown; n.d."not determined.
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
1
2
3
4
5
6
7
8
Identi"cation(H tentative)
1000
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
PC. The 8-hydroxylated #avonoids 12, 14}16 were previously isolated from two
species of Lavandula, L. coronopifolia and L. pubescens, and identi"ed by means of
NMR, MS and UV spectroscopy as the 8-glucuronides of hypolaetin, hypolaetin
4@-methyl ether and isoscutellarein and the 7-glucoside of hypolaetin, respectively (El
Garf et al., 1999). The compounds detected and identi"ed (sometimes tentatively)
during the combined HPLC, PC and electrophoresis analyses are presented in Table
3. The external #avonoids 18}21 (see Table 2) may be present in a larger quantity and
in more species than is shown in Table 3, as the methods of extraction and the HPLC
programmes were more suitable for #avonoid glycosides than for external #avones.
Absorbance of the peaks at the j
of each #avonoid was used as a measure of the
.!9
relative amounts (see Table 3). The solvent systems used for HPLC, which contained
a small percentage of HOAc, did not distinguish between glucosides and glucuronides
of the same aglycone, as these had more or less the same retention times. However, the
presence of both 1 and 2, and both 4 and 5 in all taxa investigated in sections Stoechas,
Dentata and Lavandula was con"rmed by means of paper electrophoresis of the
extracts and 2-D paper chromatography in BAW and water. Flavonoid glucuronides
are very mobile in water compared with the corresponding monoglucosides and so the
two are easily separated in this system.
A number of accessions were investigated by means of 2-D paper chromatography
only. As this technique is generally less sensitive than HPLC, only presence of the
major #avonoids 1, 2, 4 and 5 and the 8-hydroxy#avone glycosides 12 and 16 could be
scored. The latter compounds are easy to detect on paper chromatograms because
they do not change colour in UV light after fuming with ammonia. Moreover, they
have very characteristic UV spectra. These results are presented in Table 4 and largely
agree with the "ndings of the more detailed analyses presented in Table 3.
The #avonoid pro"les observed on the 2-D chromatograms and HPLC traces
showed very little variation among di!erent accessions, subspecies and varieties of the
same species. Moreover, there was surprisingly little variation in major #avonoid
pro"les among species belonging to the same section, any di!erences being mainly
quantitative. Section Lavandula appeared to be the most uniform group in that all
members produced #avone monoglycosides 1}6, #avone diglycosides 7 and 8, and the
aglycones, apigenin (18) and genkwanin (19). Again, in section Stoechas the di!erences
between the three taxa were very minor. However, in section Pterostoechas only two of
the seven taxa fully investigated (Table 3) had identical #avonoid pro"les, namely L.
maroccana and L. rotundifolia, although in all seven species 8-hydroxy#avone glycosides were the major #avonoid constituents. Lavandula mairei var. mairei di!ered from
all other members of this group in the presence of a chrysoeriol 7-glycoside (3) and
apigenin glycosides (4 and 5) and the absence of 8-hydroxy#avone glycosides 12}15.
Again, L. minutolii var. minutolii was distinguished by the absence of simple #avone
glycosides and the presence of #avone aglycones. Within section Subnudae, L. aristibracteata and L. subnuda have almost identical leaf #avonoid pro"les with the absence
of the simple #avone glycosides found in L. dhofarensis and L. nimmoi.
However, at sectional level profound di!erences were found in that the distribution
of the di!erent #avonoid classes, i.e. #avone glycosides, #avone diglycosides, 6hydroxy#avone glycosides, 8-hydroxy#avone glycosides and #avone C-glycosides,
Table 3
Presence of #avonoids in species, subspecies and varieties of Lavandula and Sabaudia based on HPLC, PC and electrophoresis results%
8-OH Flavone
8- glycosides
7/8
9
10
11
12
13
14
15
16
17
18
19
20
21
## ## ## $
## ! ## $
## ## ## #
!
!
!
!
!
!
!
!
!
$
$
$
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
$
$
$
$
$
$
!
!
!
!
$
$
L. dentata var. dentata%
Section Lavandula
#
!
#
$
!
$
## ## !
!
!
!
!
!
$
$
!
!
L. angustifolia subsp. angustifolia%
L. angustifolia subsp. pyrenaica
L. angustifolia subsp. delphinensis
L. lanata%
L. latifolia%
Section Pterostoechas
## $
## $
## $
## $
## $
#
#
#
#
#
$
$
$
$
$
$
$
$
$
$
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
$
$
$
$
$
$
$
$
$
$
!
!
!
!
!
!
!
!
!
!
L. canariensis%
$
!
!
!
!
!
!
!
## #
!
#
$
#
!
!
!
!
L.
L.
L.
L.
L.
L.
$
$
$
!
$
$
!
!
$
!
!
!
!
!
!
!
!
!
!
!
$
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
## ## !
## ## $
! ! !
## ! !
## ! !
## ## #
## ## !
## ## #
! # #
# $ #
# ## $
## ## #
!
!
!
#
!
!
!
!
!
!
!
!
!
!
#
#
!
!
!
!
!
$
!
!
Compound
Texon
Section Stoechas
L. stoechas subsp. stoechas%
L. stoechas subsp. luiseiri
L. viridis%
Section Dentata
coronopifolia
maroccana%
mairei var. mairei%
minutolii var. minutolii%
multixda
rotundifolia
1/2
3
4/5
6
1001
2continued
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
Flavone aglycones
8-OH
Flavone 7- (external)
glycosides
Fla.
6-OH
C-gly.
Flavone
glycosides
Flavone glycosides
1002
Compound
Texon
Flavone aglycones
8-OH
Flavone 7- (external)
glycosides
Fla. 8-OH Flavone
6-OH
C-gly. 8- glycosides
Flavone
glycosides
Flavone glycosides
1/2
3
4/5
6
7/8
9
10
11
12
Section Subnuda
L. aristibracteata%
L. dhofarensis%
L. subnuda
!
$
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
Section Chaetostachys
L. bipinnata%
$
!
!
!
!
!
!
Sabaudia group
S. atriplicifolia
$
!
$
#
!
!
!
13
17
18
19
20
21
# ## # $ $
## # ## ! $
## # # ## $
$
!
$
!
!
!
!
!
!
!
!
!
#
!
!
!
## ## #
$
$
$
!
!
!
$
!
!
!
#
## !
!
!
!
!
14
!
15
16
%Extract subjected to electrophoresis; Fla. C-Gly.: Flavone C-glycoside; !Flavonoid not detected by HPLC; $, #, ## Relative amounts of #avonoids
present (low- medium- or high-UV absorbance of the #avonoid peak, respectively).
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
Table 3*continued
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
1003
Table 4
The distribution of #avonoids in additional species, subspecies and varieties of Lavandula and Sabaudia
based on PC results only
Taxon
Flavone glycosides
8-OH Flavone glycosides
1
2
4
5
12
16
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
!
!
!
!
!
!
!
!
!
!
Section Dentata
L. dentata var. candicans
#
#
#
#
!
!
Section Pterostoechas
L. antineae
L. buchii var. buchii
L. buchii var. gracile
L. mairei var. antiatlantica
L. minutolii var. tenuipinna
L. pinnata
L. tenuisecta
$
$
$
$
!
$
$
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
#
#
#
!
#
#
#
#
#
#
#
#
#
#
Section Subnudae
L. nimmoi
$
!
!
!
#
#
Sabaudia group
S. erythreae
#
#
!
!
!
#
Section Stoechas
L. stoechas subsp.
L. stoechas subsp.
L. stoechas subsp.
L. stoechas subsp.
L. stoechas subsp.
atlantica
cariensis
lusitanica
pedunculata
sampaiana
was restricted to certain sections. For example, the #avone diglycosides, luteolin
7,4@-diglucuronide (7) and 7-glucoside-4@-glucuronide (8) were found only in section
Lavandula and the #avone C-glycoside, vitexin (11), only in sections Stoechas and
Dentata. 8-Hydroxy#avone glycosides occurred widely in sections Pterostoechas,
Subnuda and Chaetostachys and in Sabaudia, whereas 6-hydroxy#avone glycosides
were restricted to section Dentata. As to leaf surface #avonoids, apigenin (18) and
genkwanin (19) were regularly detected in members of sections Stoechas, Dentata and
Lavandula, whereas the highly methoxylated external #avones 20 and 21 were found in
sections Stoechas, Pterostoechas, Subnudae and Chaetostachys.
4. Discussion
The combined use of 2-D paper chromatography and HPLC analysis has provided
both a detailed and comprehensive survey of the leaf #avonoids in Lavandula. The
1004
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
results show a strong correlation between the accumulation of certain #avonoid
classes and groupings of species, which is congruent with the present sectional
classi"cation. The variation found at the lower taxonomic ranks was either not great
enough to be informative or appeared to be random and not correlated to any
taxonomic entity presently recognised. However, #avonoid patterns do appear to be
of systematic use at the sectional level and above in Lavandula. The distribution of the
di!erent #avonoid classes in the sections of Lavandula and in Sabaudia is presented in
Table 5. In this Table accumulation rather than presence of a #avonoid class was often
taken as a character, because chemotaxonomically accumulation of a chemical feature
is more important than mere presence. The sensitivity of HPLC can reveal presence of
trace compounds which serve as precursors of the ones that accumulate. For instance,
small amounts of a #avone 7-monoglycoside were present in section Pterostoechas,
Subnuda and Chaetostachys, but only 8-hydroxylated #avone glycosides were accumulated in these taxa.
Three major groupings of taxa can be identi"ed using #avonoid features as
characters. The "rst group corresponds to sections Stoechas, Dentata and Lavandula,
which are characterised by the accumulation of #avone 7-monoglycosides. However,
each of these sections can be distinguished by its overall #avonoid pro"le (see Table 5).
Thus, Lavandula is unique in producing #avonoid diglycosides, whereas sections
Dentata and Stoechas both contain #avone C-glycosides. But section Dentata di!ers
from section Stoechas in the presence of 6-hydroxylated #avone 7-glycosides. The
second chemical group within the genus contains sections Pterostoechas, Subnuda and
Chaetostachys, which are characterised by an accumulation of 8-hydroxylated #avone
7-and 8-glycosides. There are small individual di!erences among the #avonoid pro"les of the species in these sections, but no overall distinction in #avonoid patterns
among the three sections.
The third group corresponds to those taxa assigned to the genus Sabaudia, which
accumulate both 8-hydroxylated #avone 7-glycosides and #avone 7-glycosides. It is
therefore characterised by the presence of compounds which otherwise are accumulated only in group one or group two. Flavone 7-glycosides are the most common type
of #avonoid in the Lamiaceae, and therefore are not very informative about relationships within this family (TomaH s-BarberaH n et al., 1988). 8-Hydroxylated #avone glycosides, on the other hand, are uncommon in the family. Indeed, in subfamily
Nepetoideae they have only been found in the genus Lavandula (El Garf et al., 1999),
so that the presence of this class of #avonoid in Sabaudia is a very strong indication
that this taxon is very closely related to Lavandula, at least to those sections that
contain 8-hydroxy#avone glycosides. It is possible that Sabaudia is basal to all
sections of Lavandula, but that the species of sections Stoechas, Dentata and Lavandula
have all without exception lost the ability to synthesise the 8-hydroxy#avones.
However, this does not seem very likely, as the ability to produce these #avonoids has
not been lost in any of the taxa belonging to the other three sections. It seems more
probable that a predecessor of Sabaudia has evolved the ability to produce 8hydroxy#avone 7-glycosides and that this taxon is also basal to sections Pterostoechas, Subnuda and Chaetostachys, but not to the other three sections of Lavandula.
If this is the case, the immediate predecessor of Pterostoechas, Subnuda and Chaetos-
Taxon
Accumulation of
#avone 7-Omonoglycosides
(Compounds 1}6)
Presence of
#avone di-Oglycosides
(Compounds 7
and 8)
Accumulation of
6-hydroxylated
#avone 7-Oglycosides (Compounds 9 and 10)
Presence of
#avone C-glycosides (Compound
11)
Accumulation
of 8-hydroxylated
#avone 8-Oglycosides
(Compounds
12}15)
Accumulation of
8-hydroxylated
#avone 7-Oglycosides
(Compounds
16 and 17)
Presence of
external #avones
(Compounds
18}21)
Section Stoechas
Section Dentata
Section Lavandula
Section Pterostoechas
Section Subnudae
Section Chaetostachys
Sabaudia group
#
#
#
!
!
!
#
!
!
#
!
!
!
!
!
#
!
!
!
!
!
#
#
!
!
!
!
!
!
!
!
#
#
#
!
!
!
!
#
#
#
#
#
#
#
#
#
#
?
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
Table 5
The distribution of di!erent #avonoid classes in sections of Lavandula and in Sabaudia
1005
1006
T.M. Upson et al. / Biochemical Systematics and Ecology 28 (2000) 991}1007
tachys must also have evolved separately the ability to produce 8-hydroxy#avones
8-glycosides.
The major groupings of sections suggested by the #avonoid data are highly
congruent with the major groupings suggested by other data sets such as gross
morphology, carpology, molecular sequence data and palynology (Upson, 1997). At
present, this grouping of sections is not re#ected in any classi"cations of the genus, but
evidently these data need to be incorporated. The results obtained here provide
further compelling evidence to support the recognition of these groupings. The rank at
which to recognise these groups is a more complex question and in the taxonomic
hierarchy they probably represent either subgenera or even distinct generic groupings.
Acknowledgements
The University of Reading is gratefully acknowledged for funding to T.M.U.
through a postgraduate scholarship. We thank the following people for their help in
supplying plant material for use in this study: J. Head (National Plant Collection
holder of Lavandula under the National Council for the Conservation of Plants and
Gardens Scheme), W. Lobin (Botanischer Garten der UniversitaK t Bonn), A.G. Miller
(Royal Botanic Garden, Edinburgh), H.D.V. Prendergast (Royal Botanic Gardens,
Kew), N.J. Singh (Botanical Survey of India) and the Curators of the following
herbaria: BM, C, E, M and O. Thanks to Dr. Stephen Jury of the University of
Reading for help and advice in undertaking this work.
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