Biochemical Systematics and Ecology 37 (2009) 285–289

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Biochemical Systematics and Ecology

journal homepage: www.elsevier.com/locate/biochemsyseco

Iridoid and caffeoyl phenylethanoid glycosides of the endangered

carnivorous plant Pinguicula lusitanica L. (Lentibulariaceae)
Tomás Grevenstuk a, Justin J.J. van der Hooft b, Jacques Vervoort b, c, Pieter de Waard c,
Anabela Romano a, *
a
IBB-Institute for Biotechnology and Bioengeneering, Faculty of Sciences and Technology, University of Algarve,
Campus of Gambelas, Ed. 8, 8005-139 Faro, Portugal
b
Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands
c
Wageningen NMR Center, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: This work reports for the first time the identification of the major compounds of Pinguicula
Received 7 January 2009 lusitanica, an endangered carnivorous plant species, using minimal amounts of plant
Accepted 2 May 2009 material. A methanol extract was prepared from in vitro cultured plantlets and analyzed by
HPLC–SPE–NMR/HPLC–MS. Three iridoid and five caffeoyl phenylethanoid glycosides were
Keywords: identified. These groups of natural compounds were previously reported in the Lentibu-
Lamiales
lariaceae family and have been used as chemotaxonomic markers in related families.
Globularin
Ó 2009 Elsevier Ltd. All rights reserved.
Verbascoside
Hyphenated techniques
LC–MS
HPLC–SPE–NMR

1. Introduction

Pinguicula lusitanica L., commonly known as the Pale butterwort, is a small rare carnivorous perennial plant with phar-
macological value and limited reproductive capacity. The plant grows wild in areas along coastal western Europe from
Scotland to Iberia and Morocco in north-western Africa (Heslop-Harrison, 2004). The butterworts constitute the second most
diverse genus of the carnivorous Lentibulariaceae, with 85 currently accepted species (Cieslak et al., 2005).
Wild populations of this species are becoming increasingly scarce, probably due to the fact that reproduction of P. lusi-
tanica relies entirely on seed production. Moreover, seedling establishment in a suitable environment is crucial because
seedlings do not withstand competition (Heslop-Harrison, 2004). An efficient micropropagation protocol for P. lusitanica has
been published recently (Gonçalves et al., 2008), which enabled this chemical study, as the natural populations cannot
provide sufficient material for analysis due to their reduced number of individuals.
There are no reports on the biochemical or physiological data of P. lusitanica. In fact, only two of the some 85 species of the
Pinguicula genus have been subject of a biochemical study, namely Pinguicula vulgaris (Damtoft et al., 1985; Marco, 1985) and
Pinguicula moranensis (Damtoft et al., 1994). Several iridoid glucosides were identified in these two species, and a caffeoyl
phenylethanoid glycoside, namely verbascoside, was found in P. moranensis as well. These groups of compounds are char-
acteristic for the Lentibulariaceae and related families (Order Lamiales), and have proven to be good chemotaxonomic
markers in several related families (Rønsted et al., 2003; Jensen et al., 2005; Taskova et al., 2005; Pedersen et al., 2007).

* Corresponding author. Tel.: þ351 289800910; fax: þ351 289818419.

E-mail address: aromano@ualg.pt (A. Romano).

0305-1978/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bse.2009.05.003
286 T. Grevenstuk et al. / Biochemical Systematics and Ecology 37 (2009) 285–289

The function of phenylethanoid glycosides in plants can be resistance to, or protection from, fungal or viral attacks
(Jiménez & Riguera, 1994). The benefit of iridoid glucosides for the plant is not clear, although it has been demonstrated that
the iridoid aucubin is a strong protein denaturant when hydrolyzed by the enzymes in the plant. This observation indicates
that iridoid glucosides may play an important role in the defense mechanism against herbivores (Konno et al., 1999; Jensen
et al., 2002). To better understand the biological function of these compounds, further chemical investigation on other
Pinguicula species is needed.
The interfacing of liquid chromatography with NMR spectroscopy together with the introduction of on-line solid-phase
extraction (SPE) provides structural information directly from crude extracts, allowing to analyze small biological samples
(Albert, 2002; Clarkson et al., 2005; Exarchou et al., 2005; Jaroszewski, 2005), important in the study of endangered plant
species such as P. lusitanica.
This is the first report of the chemical screening of the major secondary metabolites of P. lusitanica by HPLC–SPE–NMR
without using field specimens for obtaining plant material.

2. Materials and methods

2.1. Plant material

In vitro plantlets of P. lusitanica were produced according to the protocol described by Gonçalves et al. (2008). The original
plant material, specimen number LISU215272, was authenticated by Dr. A. I. Correia from the Botanical Garden of the
University of Lisbon.

2.2. Sample preparation

In vitro cultured P. lusitanica plantlets (50 g) were extracted twice for 24 h with methanol (HPLC-grade, Fluka, Buchs,
Switzerland). The obtained extract was filtered (Whatman no 1, Springfield Mill, England), concentrated under rotary vacuum
evaporation and dissolved in water. After freeze drying, 760 mg of extract was obtained. A solid-phase extraction column
(SUPELCLEANÔ LC-18 Packing; 60 mL; 10 g) was used for sample preparation to remove the most apolar and fatty constit-
uents of the extracts and served as a sample cleanup. For analysis, 25 mg of cleaned extract were dissolved in 500 mL of 50%
methanol solution in water.

2.3. HPLC–MS/HPLC–SPE–NMR

The HPLC–SPE–NMR/HPLC–MS measurements were performed with a Agilent 1200 quaternary solvent delivery pump
equipped with a Spark Prospekt 2 solid-phase extraction (SPE) device, containing HySphere resin SH cartridges (10  2 mm,
25–35 mm), a Bruker Avance III 600 spectrometer, a Bruker Daltonics MicrOTOF ESI mass spectrometer and HyStar 3.2
software. Topspin 2.0 software was used for NMR experiments. The chromatographic separation was conducted with an
Alltima HP 3 mm (150  4.6 mm i.d.) column using a binary eluent consisting of water containing 0.1% (v/v) formic acid
(solvent A) and acetonitrile (solvent B), with a 0.6 mL/min flowrate. The following linear gradient was applied: at 0 min 10% B;
at 5 min 15% B; at 43 min 25% B; at 45 min 95% B; at 47 min 95% B; and at 50 min 10% B followed by a 5 min conditioning step.
The injection volume was 20 mL. The HPLC eluate was monitored by DAD, and for each compound one absorption threshold at
220 (1), 280 (2–7), or 312 (8) nm was defined in order to provide start and stop signals for the SPE trappings. A total of four
cumulative trappings were performed for each peak selected for analysis. The cartridges were dried with nitrogen gas and the
analytes were eluted with methanol-d4 to the NMR flow probe. 1H NMR spectra were recorded at 600 MHz using standard
pulse sequences delivered by Bruker. After NMR experiments, the cartridge content was flushed from the probe in to vials and
analyzed by ESI–MS. Ions were detected in negative mode in the range of m/z 100–1500. The MS working conditions were as
follows: the nebulizer pressure was 3.0 bar, the drying gas flow equalled 8.0 L/min, the drying gas temperature was 190  C,
and the capillary spray was 4.2 kV.

3. Results and discussion

The main compounds identified in this study in the P. lusitanica extract were iridoid glucosides and caffeoyl phenyl-
ethanoid glycosides. Eight peaks were separated by chromatography and selected for analysis (Fig. 1). The identified iridoid
glucosides were mussaenosidic acid (1) (Çaliş et al., 1993), scutellarioside II (4) (Çaliş et al., 1993), and scutellarioside I or
globularin (8) (Damtoft et al., 1985). Another five caffeoyl phenylethanoid glycosides, namely verbascoside or acteoside (7), R
and S campneoside II (5,6) and R and S campneoside I (2,3) were identified by comparison with literature data (Wu et al.,
2004). The chemical structures of the identified compounds are shown in Fig. 2.
Caffeoyl phenylethanoid glycosides are characteristic for most taxa in Lamiales, and the most common representative,
verbascoside, has been reported in all the families of Lamiales. Only two examples are known from outside the order (Jensen,
1992; Taskova et al., 2005). Iridoid glucosides are characteristic for Lamiales, but they are more widespread than caffeoyl
phenylethanoids. However, specific compounds can be systematically very useful. Aucubin and catalpol, for instance, are
characteristic for most taxa within Plantaginaceae (Taskova et al., 2005).
 T. Grevenstuk et al. / Biochemical Systematics and Ecology 37 (2009) 285–289 287

800 100

7
Intensity (mAU) 600 75

Acetonitrile (%)
8

400 50

200 3 25
2
56

1
0 0

0 10 20 30 40 50
Retention time (min)

Fig. 1. HPLC chromatogram monitored at 280 nm of the methanol extract of P. lusitanica (150  4.6 mm i.d. column; gradient elution profile is shown as percent
of acetonitrile in H2O). The identified secondary metabolites are labeled 1–8 (Structures in Fig. 2). Maximum absorbance of peak 1 is at 220 nm.

In Lentibulariaceae, verbascoside has been found in Pinguicula, however it was not detected in Utricularia (Damtoft et al.,
1994). As to what concerns the content in iridoid glucosides, catalpol, scutellarioside II and globularin were found in P. vulgaris
(Damtoft et al., 1985; Marco, 1985), catalpol in P. moranensis and mussaenosidic acid, aucubin and gardioside in Utricularis
australis (Damtoft et al., 1994). It is interesting to see that globularin and scutelarioside II are both present in P. lusitanica and
P. vulgaris, while the co-occurrence of these iridoid glucosides is rather restricted outside the Lamiales. The occurrence of
verbascoside and the iridoid glucosides mussaenosidic acid, scutellarioside II and globularin, corroborates that P. lusitanica is
a true member of Lamiales.

O OH HO

O
O
O
O O O
HO O O OH
OH
R O
HO OH
HO OH
OH
OH
4 Scutellarioside II R=OH
1 Mussaenosidic Acid
8 Globularin R=H

OH
HO

O OH
OH
O OH
HO O
O
O O
HO
OH R
OH

2, 3 R/S Campneoside I R=OH

5, 6 R/S Campneoside II R=OCH3

7 Acteoside R=H

Fig. 2. Structures of the main constituents of P. lusitanica.

288 T. Grevenstuk et al. / Biochemical Systematics and Ecology 37 (2009) 285–289

The intensity of the NMR signals indicates that globularin is the most abundant in the P. lusitanica extract, followed closely
by verbascoside. The relative amount of these two compounds is similar, suggesting that the pathways for these two types of
molecules in P. lusitanica are of equal importance. P. vulgaris and P. lusitanica have the same compounds in their constitution,
while being genetically considerably distinct, based on analysis of the trnK intron (Cieslak et al., 2005). This observation
suggests that the two main chemical pathways are rather conserved in the Pinguicula genus.
Mussaenosidic acid is most likely the precursor of scutellarioside II and globularin. The intensities of the NMR signals
indicate that mussaenosidic acid and globularin are the most abundant iridoids (twice as concentrated as scutellarioside II),
remaining unclear why mussaenosidic acid is produced in such high amounts, as its biological importance is unknown.
However, one can assume that scutellarioside II and globularin are part of the plant’s defense mechanism. As has been
reported for structurally related iridoids, after removal of the glycosyl group, the aglycone binds to macromolecules
decreasing the nutritive value of the dietary protein, thereby preventing herbivores from eating the plant (Konno et al., 1999).
The caffeoyl phenylethanoid glycosides are biologically active due to the catechol unit and the redoxcycling mechanism, by
which reactive oxygen species (ROS) are formed (Chichirau et al., 2008). These ROS can be damaging to cells from an attacking
organism. The glycosylated form, as well as the aglycone is biologically active, although the removal of the glycosyl unit
decreases the compounds’ polarity, enhancing the cellular membrane crossing.
Both isomers of campneoside I and II are likely derived from verbascoside after hydroxylation and methylation, respec-
tively. The intensities of the UV and the NMR signals of the two pair of isomers were identical, indicating that there is no
difference in concentration in the extract between these two molecules. This suggests that the reactions involved in the
hydroxylation (to give R and S campneoside I), and posterior methylation (to give R and S campneoside II) of acteoside are
chemical processes, or reactions without a stereochemical preference, in case we are dealing with enzymatic processes.
Chemotaxonomy, together with molecular biology techniques is of great importance in plant science, as chemotaxonomic
characters can be useful to support relationships retrieved in DNA-sequence analyses (Grayer et al., 1999; Jensen et al., 2005).
Caffeoyl phenylethanoid and iridoid glycosides have taxonomic and genetic importance and have been used to establish
taxonomic relations between species of several plant families (Bignoniaceae, Labitaeae, Oleaceae). The genera Digitalis
(Taskova et al., 2005), Veronica (Jensen et al., 2005; Pedersen et al., 2007) and Plantago (Rønsted et al., 2000, 2003) of the
Plantaginaceae family, and Rehmannia (Albach et al., 2007) of the Scrophulariaceae family, have been previously studied
phytochemically and taxonomic relations have been inferred based on these classes of natural compounds. The genus
Veronica, for instance, has been recently transferred from the Scrophulariaceae to the Plantaginaceae family based on
molecular investigations and supported by chemical findings (Pedersen et al., 2007).
In summary, the present study is the first report of the main secondary metabolites produced by P. lusitanica. These
compounds are iridoid glucosides and caffeoyl phenylethanoid glycosides, of which the main compounds are globularin and
verbascoside, respectively, in agreement with previous chemical studies performed in species of the Lentibulariaceae family.

Acknowledgements

This research was supported by the European Community activity Large-Scale Facility Wageningen NMR Center (FP6-
2004-026164 (2006–2009). T. Grevenstuk acknowledges a grant from the Portuguese Science and Technology Foundation
(FCT, Grant SFRH/BD/31777/2006).

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