The phytoecdysteroid profiles of 7 species of Silene (Caryophyllaceae).

A r t i c l e
THE PHYTOECDYSTEROID
PROFILES OF 7 SPECIES OF Silene
(CARYOPHYLLACEAE)
Larisa Zibareva, Valentina I. Yeriomina, and
Nyamjav Munkhjargal
Tomsk State University, Siberian Botanical Garden, 634050 Tomsk,
pr. Lenina, 36, Russia
Jean-Pierre Girault
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques,
CNRS UMR 8601, Universite´ Paris Descartes, 45 rue des Saints Pe`res,
75270 Paris, Cedex 06, France
Laurence Dinan and René Lafont
Laboratoire de Biochimie structurale et fonctionnelle des Prote´ines,
CNRS FRE 2852, Universite´ Pierre et Marie Curie, Case 29, 7 Quai
Saint Bernard, 75252 Paris, Cedex 05, France
The phytoecdysteroid profiles of extracts of aerial parts of flowering
plants of 7 ecdysteroid-containing species in the genus Silene
(Caryophyllaceae; S. fridvaldszkyana Hampe, S. gigantea L.,
S. graminifolia Otth, S. mellifera Boiss. & Reuter, S. repens Patr.,
S. schmuckeri Wettst., and S. sendtneri Boiss.) have been examined and
identified by HPLC and, in the case of two new compounds, by mass
spectrometry and NMR. S. frivaldszkyana was found to contain
predominantly 20-hydroxyecdysone (20E), with smaller amounts of
2-deoxyecdysone (2dE), 2-deoxy-20-hydroxyecdysone (2d20E), polypodine B (polB), integristerone A (IntA), 26-hydroxypolypodine B
(26polB), and 20,26-dihydroxyecdysone (20,26E). Additionally, a new
minor ecdysteroid, 26-hydroxyintegristerone A, has been identified from
this species. S. gigantea contains 3 major ecdysteroids (2dE, 2d20E, and
20E) and much smaller amounts of intA and 2-deoxy-20-hydroxyecdysone 25-b-D-glucoside, which is a new ecdysteroid. Ecdysteroids in the
other 5 species have been identified by co-chromatography with reference
Grant sponsors: CNRS; UPMC.
Correspondence to: Larisa Zibareva, Tomsk State University, Siberian Botanical Garden, 634050 Tomsk,
pr. Lenina, 36, Russia. E-mail: zibareval@inbox.ru
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 72, No. 4, 234–248 (2009)
Published online in Wiley InterScience (www.interscience.wiley.com).
& 2009 Wiley Periodicals, Inc. DOI: 10.1002/arch.20331
Phytoecdysteroid Profiles of 7 Species of Silene
235
compounds on RP- and NP-HPLC systems. There is considerable
variability with regard to ecdysteroid profiles within the genus Silene. The
chemotaxonomic value of ecdysteroid profiles within the genus Silene is
C 2009 Wiley Periodicals, Inc.
discussed. Keywords: chemotaxonomy; ecdysteroid; HPLC; 20-hydroxyecdysone;
NMR; phytoecdysteroid
INTRODUCTION
Phytoecdysteroids are analogues of arthropod steroid hormones occurring in plants,
where they are believed to contribute to the defence of the plant against invertebrate
predation (Dinan, 2001). Analysis of the seeds of a large number of species for the
presence of ecdysteroids has revealed that 5–6% of species of higher plants contain
these compounds (Dinan, 1995), but assessment of leaves indicates that many more
plants contain at least detectable levels of ecdysteroids during growth (Dinan et al.,
2001). In total, 4300 different ecdysteroid analogues have been identified from plants
(Lafont et al., 2002), which has proved to be very beneficial for structure-activity
studies (Dinan, 2003) and for provision of ecdysteroids (especially 20-hydroxyecdysone) for pharmaceutical and commercial preparations (Dinan and Lafont, 2006),
even if the ecdysteroids themselves have limited potential as exogenous invertebrate
pest control agents (Dinan, 1989). The apparently erratic distribution of ecdysteroids
amongst plant species was originally an enigma. However, as more species within
specific plant families have been investigated, patterns are beginning to emerge (Dinan
et al., 1998; Zibareva, 2000), and this has led to the idea that phytoecdysteroids might
lend themselves for chemotaxonomic purposes. Amongst the genera that seem most
suitable for examination of the validity of their chemotaxonomic usefulness is Silene,
since it is a large genus, which has received much attention with regard to the presence
of ecdysteroids in its constituent species. It has also been the subject of
several extensive morphological and, more recently, molecular taxonomic studies
(Chowdhuri, 1957; Greuter, 1995; Oxelman et al., 1997, 2001).
Silene is one of the richest ecdysteroid-containing genera so far detected in the
plant world. The genus contains 4700 species (Greuter, 1995). Ecdysteroids have
been recognised in more than 120 species and subspecies of the genus from the 155 so
far tested (summarised in Ecdybase; Dinan and Lafont, 2002). Commonly, the
ecdysteroid-positive species possess a rich ecdysteroid composition; high levels of
20-hydroxyecdysone (20E), accompanied by 2-deoxy-20-hydroxyecdysone, 2-deoxyecdysone, polypodine B, and integristerone A, are characteristic for many Silene
species (see Fig. 1 for structures of these compounds).
Plants of the genus Silene are characterised by the presence of not only a large
number of free ecdysteroids, but also various derivatives, including benzoates,
sulphates, and glucosides (20-hydroxyecdysone 22-O-benzoate-25-glucoside, 2-deoxypolypodine B 3b-D-glucoside, 20-hydroxyecdysone 3b-D-glucoside, ecdysteroside,
etc.; Saatov et al., 1999). Many new ecdysteroids could be isolated from members of
this genus and many of these analogues have, so far, only been found in plants of this
genus. There are certain differences in ecdysteroid variety and levels between species,
which indicates that ecdysteroid profiles could additionally be used for chemotaxonomic purposes.
Archives of Insect Biochemistry and Physiology
236
Archives of Insect Biochemistry and Physiology, December 2009
R4
OH
OH
OH
R5
OH
OH
R1
HO
OH
R3
O
Ecdysteroid
ecdysone
2-deoxyecdysone
20-hydroxyecdysone
2-deoxy-20-hydroxyecdysone
polypodine B
26-hydroxypolypodine B
20,26-dihydroxyecdysone
integristerone A
OH
OH
HO
H
HO
O
IX 26-hydroxyintegristerone A
No.
I
II
III
IV
V
VI
VII
VIII
Glc
OH
R2
HO
OH
OH
OH
R
-H
-H
-H
-H
-H
-H
-H
-OH
R
-OH
-H
-OH
-H
-OH
-OH
-OH
-OH
R
-H
-H
-H
-H
-OH
-OH
-H
-H
R
-H
-H
-OH
-OH
-OH
-OH
-OH
-OH
H
O
X 2-deoxy-20-hydroxyecdysone 25-glucoside
R
-H
-H
-H
-H
-H
-OH
-OH
-H
Figure 1. Ecdysteroid structures.
It has been shown that some Sections of Silene (for example, Siphonomorpha,
Chloranthae, Coronatae, Graminiformes, Otites, Silene, Dipterosperma, Lasiocalycinae,
Holopetalae) consist of only ecdysteroid-containing species, whereas other Sections
(Behen, Atocion, Psammophilae, Odontopetalae, etc.) contain only ecdysteroid-negative
species (Zibareva et al., 2003a). In these Sections, it is possible, with high probability,
to predict ecdysteroid presence or absence in as yet uninvestigated species.
Others Sections (Sclerocalycinae, Spergulifoliae, Saxifragoideae, Rigidulae) contain both
ecdysteroid-positive and -negative species.
Most of the various ecdysteroid analogues isolated from the genus Silene (39 in
total) have been isolated from the Section Otites; however, most of the ecdysteroidpositive species are present in the Section Siphonomorpha.
The ecdysteroid composition is characteristic for each Section (Table 1).
20-Hydroxyecdysone and polypodine B are characteristic for plants of the
Section Siphonomorpha. Ecdysone, integristerone A, and 2-deoxyintegristerone A are
additionally characteristic for the Section Silene, while, for the Section Otites,
20-hydroxyecdysone, ecdysone, 2-deoxy-20-hydroxyecdysone, 2-deoxyecdysone,
2-deoxyintegristerone A, 2-deoxy-21-hydroxyecdysone, and sidisterone are typical.
The classification of the genus Silene was altered in accord with morphological
attributes (Zibareva et al., 2003a), when compared with the classification of the Flora
Europaea (Chater et al., 1993). In particular, the Section Sclerocalycinae was divided into
four subsections (Fig. 2a): Sclerocalycinae, Chloranthae, Coronatae, Tataricae. It can be
questioned whether this was justified. There are not enough current data concerning
ecdysteroid composition to assess fully the competency of such a division. However, the
ecdysteroid composition of each of the four subsections differs. 20-Hydroxyecdysone and
polypodine B are found in plants of all subsections. Additionally, each subsection is
characterised by the presence of other distinct ecdysteroids (Fig. 2b). In the first
subsection, 2-deoxy-20-hydroxyecdysone is found; in the second, 2-deoxy-20-hydroxyecdysone and 26-derivatives; in the third, ecdysone; in the fourth, glucosides and benzoates.
In this report, we use HPLC to analyse the ecdysteroid profiles in 7 additional
species of the genus Silene. The ecdysteroids present in significant amounts in each of
the extracts are identified and two new ecdysteroids are fully characterised. The
resulting information will contribute to the assessment of the chemotaxonomic
usefulness of ecdysteroids in this genus.
Archives of Insect Biochemistry and Physiology
Phytoecdysteroid Profiles of 7 Species of Silene
237
Table 1. Chemical Shifts (ppm) of 26-Hydroxyintegristerone A in Deuterated Water (D2O)
1
H
26IntA
H-1eq
H-2ax
H-3eq
H-4ax
H-4eq
H-5
3.93
4.05
4.15
1.82
1.82
2.63
H-7
6.00 (d, 2.1)
H-9
3.04 (s, b, w1/2 5 22)
H-11ax
H-11eq
H-12ax
H-12eq
1.77
1.78
1.96
1.96
13
C
26IntA
(s, b, w1/2 5 11)
(t, 3)
(s, b, w1/2 5 9)
C-1
C-2
C-3
C-4
76.9
69.8
71.5
34.3
(ta, b, 9.2)
C-5
C-6
C-7
C-8
C-9
C-10
C-11
47.8
n.d.
123.6
n.d.
37.0
45.0
22.7
C-12
33.5
C-13
C-14
C-15
49.7
87.6
32.8
C-16
22.5
C-17
C-18
C-19
C-20
C-21
C-22
C-23
51.7
19.5
21.4
80.5
22.1
79.8
27.7
C-24
38.0
C-25
C-26
C-27
75.7
71.3
24.4
24.9
(2 epimers)
H-15a
H-15b
H-16a
H-16b
H-17
Me-18
Me-19
1.67
2.06 (m)
1.88b
1.78b
2.32 (t, 9.9)
0.88 (s)
1.10 (s, b)
Me-21
H-22
H-23a
H-23b
H-24a
H-24b
1.24 (s)
3.43 (d, 10)
1.34
1.66
1.76
1.48 (t, d, 13.4, 3.9)
26-CH2OH
Me-27
3.47
1.17 (s)
1.18 (s)
(2 epimers)
n.d., not determined. w1/2: width at half-height in Hz; s: singlet; d: doublet; t: triplet; m: multiplet; b: broad signal.
a
Deceptively simple triplet (H-4ax and H-4eq isochronous).
b
Signals can be transposed.
MATERIALS AND METHODS
Plant Material
Species (S. fridvalszkyana Hampe, S. gigantea L., S. graminifolia Otth, S. mellifera Boiss. &
Reuter, S. oligantha Boiss. & Heidr. in Boiss., S. repens Patr., S. schmuckeri Wettst., and
S. sendtneri Boiss.) were selected on the basis that their ecdysteroid profiles had not
been investigated previously and they are representatives of different ecdysteroidcontaining Sections of the genus Silene. Plants were grown in the Botanical Gardens of
the University of Tomsk from seeds provided by botanical gardens in Germany, except
for S. gramifolia, which was collected from the wild in the Russian Altai. Plants were
Archives of Insect Biochemistry and Physiology
238
Archives of Insect Biochemistry and Physiology, December 2009
Sclerocalycinae
(a)
Sclerocalycinae
Silene bupleroides
Silene chlorifolia
Silene longicalycina
Chloranthae
Silene chlorantha
Silene frivaldszkyana
Silene sussamyrica
Coronatae
Ta taricae
Silene oligantha Silene tatarica
Silene reichenbachii
Silene radicosa
Sclerocalycinae
Sclerocalycinae
2-deoxy-20E
ponasterone
(b)
Chloranthae
2-deoxy-20E
26-polypodine B
20,26-dihydroxyecdysone
26-hydroxyintegristerone A
Coronatae
ecdysone
Tataricae
sileneoside
sileneoside D
2-deoxy-20E-22benzoate
20-E-20-0benzoate
ecdysteroside
Figure 2. Distribution of Silene species (a) and the ecdysteroids they contain (b) in the section Sclerocalycinae
(according to Lazkov; Zibareva et al., 2003a).
identified by 2 or 3 botanists. Voucher specimens of 6 of the species (not S. mellifera)
are stored at the Siberian Botanical Garden, Tomsk. Aerial portions were collected
when the plants were at the flowering stage. Plant material was air-dried and ground
to a powder.
Initial Extraction
Dried and powdered plant material was extracted 5 times with 70% aq. ethanol at
551C. The combined extracts were concentrated and diluted with 2 volumes of water.
The precipitate was removed and the remaining ethanol was evaporated off. The
water fraction was extracted with hexane and then with butanol. The butanol phase
was evaporated under vacuum. In the cases of S. fridvaldszkyana (240 g d.w. plant
material), S. gigantea (300 g), and S. sendtneri (440 g), the residues from the butanol
phases were subjected to open column chromatography on SiO2 (Silica gel L 100/160
[Chemapol]; 100 g; 90 cm 2 cm i.d.), eluted with CHCl3/EtOH 9:1 v/v (1000, 700, and
1,300 mL, respectively, for each species), 4:1 v/v (1,300, 900, and 1,800 mL,
respectively), and 2:1 v/v (600, 400, and 900 mL, respectively), while collecting
50-mL fractions. Ecdysteroids were crystallised from ethyl acetate/ethanol (7:1 or
5:1, v/v) and then purified by HPLC. For the other species (20 mg–100 g d.w. plant
material), the residue from the butanol phase was analysed directly by HPLC.
HPLC
The following HPLC systems were used. Solvent system NP1: Zorbax-SIL
(250 4.6 mm; 5-mm particle size) eluted with cyclohexane/isopropanol/water (CIW,
100:40:2.5 v/v/v) at 1 mL/min; Solvent system NP2: Zorbax-SIL (250 4.6 mm;
5-mm particle size) eluted with dichloromethane/isopropanol/water (DIW,
Archives of Insect Biochemistry and Physiology
Phytoecdysteroid Profiles of 7 Species of Silene
239
125:30:1.5 v/v/v) at 2 mL/min; Solvent system NP3: Zorbax-SIL (250 4.6 mm; 5-mm
particle size) eluted with dichloromethane/isopropanol/water (125:40:3 v/v/v) at 2 mL/
min; Solvent system RP1: ACE analytical column (150 4.6 mm, 5-mm particle size),
linear gradient from 15–35% ACN in H2O over 40 min at 1 mL/min; Solvent system
RP2: Phenomenex ODS-2 column (15 cm), eluted isocratically with 40% methanol in
H2O at 1 mL/min.
Isolation of Ecdysteroids
The ethanolic extracts of S. fridvaldszkyana and S. gigantea were partially purified by opencolumn chromatography on SiO2 eluted with increasing proportions of EtOH in CHCl3.
To isolate the 26IntA from S. fridvaldszkyana, the most polar fractions from SiO2
open-column chromatography (fractions 45–50) were subjected to NP-HPLC on a
semi-prep Zorbax-SIL column (1/200 ) (solvent DIW 125:40:3 v/v/v, 4 mL/min). With this
system, 20E eluted at 21.5 min, 26OHPolB at 39.5 min, 2026E at 54.5 min, and 26IntA
at 83 min. The major peaks were 26polB and 2026E. The purity of 26IntA was then
verified by RTP-HPLC on a Spherisorb ODS column, eluted isocratically with 30%
MeOH. It eluted at 4.0 min (compare 2026E and 26PolB at 6.8–6.9 min and 20E at
12.1 min). The isolated amount of 26IntA was estimated from the HPLC peak area.
For S. gigantea, fractions 29–40 were sequentially fractionated on two NP-systems:
a semi-prep. column eluted with DIW (125:40:3 v/v/v) at 4 mL/min (2d20E25G elutes
at 51 min) and then an analytical column eluted with CIW (100:50:4 v/v/v) at 1 mL/min
(2d20E25G elutes at 25.2 min).
Spectroscopic Methods
UV spectroscopy. Ecdysteroids were dissolved in absolute ethanol and UV spectra were
recorded with a Varian DMS 100 spectrometer.
Mass spectrometry. Mass spectra were recorded on a Jeol JMS-700 spectrometer either in
desorption/chemical ionisation (CI/D) mode with ammonia as the reagent gas or fastatom bombardment mode (FAB).
Nuclear Magnetic Resonance spectroscopy. NMR spectra were obtained on a Bruker
Avance500 at 300 K. The samples were lyophilised and dissolved in D2O. TSPd4,
3-(trimethylsilyl) [2,2,3,3-d4] propionic acid, sodium salt, was used as internal reference
for proton and carbon shifts (d70.2 ppm). When dissolved in CD3OD, proton and
carbon shifts (d70.2 ppm) are referenced from 1H and 13C signals of residual
CHD2OD with d CH 5 3.31 ppm and for 13C d CH 5 49.0 ppm. Chemical shifts are
expressed in ppm. 1D 1H and 13C spectra and 2D COSY, TOCSY, PFG-HSQC, and
PFG-HMBC NMR spectra further allowed the 1H and 13C assignments. Usually 50 mg
were sufficient for a complete analysis (Girault and Lafont, 1988; Girault, 1998).
Identification of Ecdysteroids by Co-Chromatography
Powdered dry plant material (2 g) was extracted 5 times with 25-mL portions of EtOH/
H2O (7:3 v/v) at 551C for 3 h. The extracts for each sample were pooled and rotary
evaporated. The residue was resuspended in 25 mL H2O and partitioned first against
hexane (25 mL: 1 ) and then against BuOH (25 mL: 2 ). The combined BuOH
phases were rotary evaporated, resuspended in 4 mL MeOH, and then centrifuged.
Archives of Insect Biochemistry and Physiology
240
Archives of Insect Biochemistry and Physiology, December 2009
Aliquots (1 mL) of each extract were separated by preparative C18-HPLC (YMC
column; 5-mm particle size; eluted with 40% aq. MeOH at 10 mL/min and monitored at
265 nm). All significant UV-absorbing peaks were collected separately and, where
possible, identified in a preliminary way by reference to the retention times of E, 20E
(1polB), 2dE, 2d20E, and IntA in the same system. Collected fractions were
evaporated. Putative identifications were confirmed or disproved by co-chromatography and co-injection of portions of the collected materials with reference standards
in an analytical RP-system (RP2) and an analytical NP-system (NP2 or NP3).
RESULTS
Silene frivaldszkyana
Preliminary analyses by TLC, ecdysteroid-specific RIA, and bioassay for ecdysteroid
agonist activity had all indicated the presence of ecdysteroids in both flowers and seeds
of S. frivaldszkyana (Zibareva et al., 2007), such that flowers (27.6 mg E eq/g d.w. with
the DBL-1 antiserum) contained far higher levels than seeds (0.11 mg E eq/g d.w.).
Normal-phase chromatography of the extract of S. fridvalszkyana (Fig. 3) revealed
the presence of at least 7 ecdysteroids, each of which could be identified by
co-chromatography in at least 2 HPLC systems. The major ecdysteroid in this extract is
20E (57% of the total identified ecdysteroids, based on peak area at 254 nm), with
lesser amounts of 2dE (15%), 2d20E (7%), polB (9%), IntA (3%), 26polB (1%),
and 20,26E (8%). In addition, there was a small amount of a polar ecdysteroid
(0.5%), eluting after 20,26E, which could be identified as 26-hydroxyintegristerone A
(Fig. 1). This compound was purified from the extract by open column chromatography on SiO2, semi-prep. NP-HPLC (Zorbax SIL column, eluted with DIW
125:40:3 v/v/v) and RP-HPLC (30% MeOH isocratic).
26-Hydroxyintegristerone A; ca. 0.6 mg; UV lmax in EtOH: 242 nm; MS (Fig. 4)
m/z 530, 512 (100%), 494, 477, 459, 378, 360; 1H and 13C NMR see Table 1.
Figure 3. NP-HPLC of extracts of Silene frivaldszkyana and Silene gigantea (Zorbax-Sil column eluted with
dichloromethane-isopropanol-water 100:40:3 v/v/v at 2 ml/min).
Archives of Insect Biochemistry and Physiology
Phytoecdysteroid Profiles of 7 Species of Silene
241
Figure 4. Chemical ionization/desorption mass spectra of (A) Compound SF7.5 (26-hydroxyintegristerone
A; MW 5 512) isolated from Silene frivaldszkyana and (B) Compound SG13 (2-deoxy-20-hydroxyecdysone
25-glucoside; MW 5 626) from S. gigantea. The signals at m/z 530 in SF7.5 and 644 in SG13 correspond to
[M1NH4]1.
The general features of the NMR of the compound (Table 2) identify it as an
unconjugated ecdysteroid with 27 C-atoms (compare spectra of ecdysone and
20-hydroxyecdysone; Girault and Lafont, 1988). Comparison with the 1H and
13
C NMR spectra for 20E reveals changes in the A-ring and side-chain, both
compatible with the presence of extra hydroxyl groups, since the 13C signals for C-1
and C-26 are significantly shifted to higher frequencies (from 38.09 ppm to 76.9 ppm
and from 30.10 ppm to 71.3 ppm, respectively). This is supported by the absence of
one of the H-1 signals and the higher frequency shift in the remaining H-1 signal (to
3.93 ppm), together with the absence of a methyl signal and the appearance
of–CH2OH signal (3.47 ppm) in 1H NMR. Further, analysis of the signals for H-1 and
C-1 and comparison to those of integristerone A (Lafont et al., 2002) show that the
hydroxyl at C-1 has a b-orientation, just as in integristerone A itself. The presence of 2
signals for Me-27 in both 1H and 13C spectra demonstrate the presence of 2 epimers
(R/S) in the proportion of approximately 4:1.
Silene gigantea
Previous TLC and RIA analyses of aerial parts of plants of S. gigantea at the fruiting
stage had indicated the presence of ecdysteroids (DBL-1: 13.3 mg E eq/g d.w.; Zibareva
et al., 2007)
The extract of this species (Fig. 3) contained 3 major ecdysteroids (20E [36% of
total of identified ecdysteroids], 2d20E [32%], and 2dE [27%]), with much smaller
amounts of IntA [3.5%] and a new ecdysteroid (2d20E25G [2%]; see Fig. 1 for
structure).
2-Deoxy-20-hydroxyecdysone 25-glucoside; ca. 0.5 mg; UV lmax in EtOH:
242 nm; MS (Fig. 4); 1H and 13C NMR see Table 2.
The 13C NMR spectrum of the new compound reveals the presence of 33 C-atoms,
compatible with an ecdysteroid conjugated to a hexose unit. Comparison with the
NMR spectra of 20E (Girault and Lafont, 1988) indicates modifications at C-2 and
C-25 relative to this compound.
This compound could be identified as 2-deoxy-20-hydroxyecdysone 25-b-Dglucoside on the basis of the following evidence. 1H NMR spectra show 5 singlet
methyl signals, so this compound belongs to the 20-hydroxyecdysone series.
Comparison of the steroid nucleus NMR data of this compound with those of
Archives of Insect Biochemistry and Physiology
242
Archives of Insect Biochemistry and Physiology, December 2009
Table 2. Chemical Shifts (ppm) of 2-Deoxy-20-Hydroxyecdysone 25-Glucoside in Deuterated Water
(D2O) and Deuterated Methanol (CD3OD)
1
D2O
CD3OD
13
1.35
1.68
1.87
1.69
4.14 (m, b, w1/2 5 18)
1.63
1.76
2.42 (d, b, 12)
1.36
1.61
1.83
1.70
3.99
1.59
1.85
2.43 (d, d, 12.5, 4.2)
5.99 (d, 2.4)
5.81 (d, 2.1)
3.18 (m, b, w1/2 5 26)
3.15 (m)
1.69a
1.82a
1.97
1.97
1.70a
1.74a
2.12a
1.82a
H
H-1ax
H-1eq
H-2ax
H-2eq
H-3eq
H-4ax
H-4eq
H-5
–
H-7
–
H-9ax
–
H-11ax
H-11eq
H-12ax
H-12eq
–
–
H-15a
H-15b
H-16a
H-16b
H-17
Me-18
Me-19
–
Me-21
H-22
H-23a
H-23b
H-24a
H-24b
–
Me-26
Me-27
H-10
H-20
H-30
H-40
H-50
H-60 a
H-60 b
1.67
2.09
1.81a
1.91a
2.35 (t, 9.5)
0.88 (s)
1.00 (s)
1.61
1.98
1.76a
1.98a
2.40 (m)
0.89 (s)
0.96 (s)
1.25 (s)
3.44 (d, 9.7)
1.73
1.39
1.86
1.61
1.19 (s)
3.36 (d)
1.75
1.46
1.96
1.51
1.30
1.30
4.67
3.23
3.53
3.37
3.48
3.92
3.70
1.27
1.28
4.46
3.17
3.36
3.24
3.26
3.88
3.65
(s)
(s)
(d, 8.1)
(d, d, 9.3, 8.1)
(t, 9.3)
(t, 9.3)
(m)
(d, d, 12.5, 2.3)
(d, d, 12.5, 6.6)
(s)
(s)
(d, 7.9)
(d, d, 9.5, 8.1)
(d, d, 11.6, 1.8)
C
D2O
CD3OD
C-1
29.3
29.5
C-2
28.1
28.7
C-3
C-4
65.8
32.7
65.3
33.0
C-5
C-6
C-7
C-8
C-9
C-10
C-11
52.3
n.d.
122.0
n.d.
36.7
37.7
21.3
52.2
n.d.
121.9
n.d.
37.6
37.6 (q)
21.4
C-12
32.3
32.6
C-13
C-14
C-15
48.9
86.8
31.3
49.1 (q)
85.6 (q)
32.6
C-16
21.3
21.4
C-17
C-18
C-19
C-20
C-21
C-22
C-23
50.3
18.2
24.2
78.2
20.6
78.2
26.8
50.3
17.8
24.2
77.6
20.8
78.5
26.5
C-24
39.7
39.8
C-25
C-26
C-27
C-10
C-20
C-30
C-40
C-50
C-60
80.9
26.9
26.9
97.8
74.6
77.1
71.2
77.1
62.3
78.6
27.2
27.2
98.5
75.0
78.0
71.7
77.6
62.8
n.d., not determined; b: broad. w1/2: width at half-height in Hz; s: singlet; d: doublet; t: triplet; m: multiplet;
b: broad signal.
a
Signals can be transposed.
2-deoxy-20E (Lafont et al., 2002) shows that they are in perfect agreement and,
consequently, this compound is a 2-deoxyecdysteroid. The presence of a sugar is
deduced straightforwardly, since one observes additional peaks in the region of
hydrogen bound to oxygenated carbons (3.2–4.7 ppm) in the 1H NMR spectrum, and
Archives of Insect Biochemistry and Physiology
Phytoecdysteroid Profiles of 7 Species of Silene
243
in the 13C NMR spectrum there are signals for the corresponding carbons
(60–100 ppm). This is in agreement with the link of the ecdysteroid with a glycoside
group. The nature and attachment of this glycoside could be determined by following
the general strategy for the identification of ecdysteroid glycosides (Maria et al., 2005).
A 25-glycosidic link is established thanks to the large 13C change (ca. 7 ppm) in the
chemical shift observed for C-25 in respect to the corresponding chemical shift of the
non-conjugated ecdysteroid. Moreover, one can observe in HMBC a correlation from
the H-10 -C-25 (d C-25 5 80.9 ppm). The identity of the sugar was determined by
means of careful examination of 1H–1H coupling patterns observed in 1H NMR. This
compound presents only large 3J Haxial–Haxial coupling constants (8–9 Hz), in
agreement with only a b-glucopyranoside structure. As the compound was obtained
from a plant, a b-D-configuration is the most probable.
This is a new ecdysteroid, but closely related analogues have been previously
identified. 20-Hydroxyecdysone 25-b-D-glucoside was isolated from Pfaffia iresinoides
(Nishimoto et al., 1988) and 2-deoxyecdysone 25-b-D-rhamnoside has recently been
isolated from the fern Microsorum membranifolium (Ho et al., 2008).
Other Species
Chromatographic separations of the extracts of the other 5 species of Silene on a
RP- and two NP-systems are presented in Figure 5 and summarised with reference to
ecdysteroids commonly found in many Silene species (20E, polB, E, 2dE, 2d20E, and
IntA) in the following text.
Silene graminifolia
There are 4 literature reports indicating the presence of ecdysteroids in S. graminifolia
(Revina et al., 1988; Saatov et al., 1993; Zibareva, 1997; Zibareva et al., 2007 [DBL-1:
1.3 mg E eq/g d.w.]). The presence of 20E and 2dE could be confirmed in the extract
analysed here by analytical HPLC.
Silene mellifera
Preliminary analyses of S. mellifera by TLC, RIA, and bioassay had indicated the
presence of ecdysteroids in seeds (0.11 mg E eq/g d.w.) and aerial parts of flowering
plants (5.2 mg E eq/g d.w.) (Zibareva et al., 2007).
With this extract, the presence of 20E and polB (in the ratio of 5.7:1) was
confirmed by analytical RP- and NP-HPLC. The presence of IntA, indicated by prep
RP-HPLC, was not supported by further analytical HPLC studies.
Silene repens
RP- and NP-HPLC analyses confirmed the presence of 20E and polB (in the ratio of
2:1) and IntA. The presence of 2d20E, indicated by prep RP-HPLC, was not supported
by analytical HPLC.
Silene schmuckeri
Previous TLC analysis of S. schmuckeri had indicated the presence of 0.3% w/w 20E of
the dry weight of the plant (Zibareva et al., 2007). For this extract, the presence of 20E
and polB (in the ratio of 5.85:1) was confirmed by RP- and NP-HPLC. The presence of
IntA, indicated by prep RP-HPLC, was not supported by further analytical HPLC.
Archives of Insect Biochemistry and Physiology
244
Archives of Insect Biochemistry and Physiology, December 2009
RP
DIW
CIW
Figure 5. RP- and NP-HPLC chromatograms for Silene extracts (S. graminifolia, S. mellifera, S. repens,
S. schmuckeri, and S. sendtneri). The left-hand column shows reversed-phase (System RP1) chromatograms of
the extracts, the middle column presents normal-phase separations on System NP1, and the right-hand
column gives normal-phase separations on System NP2.
Archives of Insect Biochemistry and Physiology
Phytoecdysteroid Profiles of 7 Species of Silene
245
Silene sendtneri
Previous TLC, RIA, and bioassay analyses of S. sendtneri had indicated the presence of
ecdysteroids in flowers (DBL-1: 17.5 mg E eq/g d.w.) and seeds (DBL-1: 0.27 mg E eq/g
d.w.) (Zibareva et al., 2007).
RP- and NP-HPLC chromatography of this extract confirmed the presence of 20E
and polB (in the ratio of 2.33:1). On normal-phase, the material co-chromatographing
with 2d20E on RP gave a small peak eluting at the Rt of 2d20E and a much larger peak
(12 ) with a retention time appropriate for 2-deoxy-5b, 20-dihydroxyecdysone.
Similarly, the peak co-chromatographing with E on RP separated on NP to give
approximately equal amounts of E and putative 5b-hydroxyecdysone. The identities of
these 5b-hydroxycompounds should be verified by further physico-chemical methods.
DISCUSSION
The genus Silene has proven to be a rich source of new and structurally varied
ecdysteroid analogues over the years, and as further species in the genus are
investigated this situation continues. Thus, it was to be expected that further new
analogues could be identified during the course of this study. In addition to a number
of previously known analogues, we have identified 26-hydroxyintegristerone A and
2-deoxy-20-hydroxyecdysone 25-glucoside as new minor ecdysteroids from
S. frivaldszkyana and S. gigantea, respectively. Both of these compounds represent
new permutations of the ecdysteroid structural modifications already found in this
genus. It is this variety of structural modifications across the members of the genus
Silene, together with the wealth of analogues and the significant proportion of species
that are ecdysteroid-positive, which makes the genus so appropriate for the assessment
of ecdysteroids as chemotaxonomic markers. In this study, the 7 species were selected
on the basis that they (1) were all believed to be ecdysteroid-positive, (2) have not been
extensively studied previously, and (3) together represent a wide distribution within
the understood taxonomical structure of the genus. To minimise variation owing to
developmental stage, all samples were harvested at the same stage of development
(mature flowering plants) and aerial parts were extracted. It is to be expected that
different portions of the plant of each species would differ somewhat both
quantitatively and qualitatively in the levels and profiles of ecdysteroids present.
Thus, marked differences are often observed between the ecdysteroid levels and
profiles of over- and underground portions of the same species (e.g., in Ajuga reptans,
Tomás et al., 1992; and Cyanotis longifolia, see Crouzet et al., pages, this issue).
Ecdysteroid levels in Silene species also vary between plant parts and fluctuate during
development (Zibareva and Yeryomina, 1996; Zibareva, 2000).
We have previously considered the relationship between taxonomy and the
presence or absence of ecdysteroids in Silene (Zibareva, 2000), and in the context of the
Caryophyllaceae (Zibareva et al., 2003b), at the species level. The species analysed here
were selected because preliminary studies and/or previous literature reports indicated
that they were ecdysteroid-containing. The seven species contained significant
amounts of ecdysteroids and it was possible to identify at least some of the analogues
present in each. In the case of S. frivaldszkyana and S. gigantea, owing to the larger
amounts of plant material extracted, it was possible to identify major and minor
ecdysteroids present, while S. graminifolia, S. mellifera, S. repens, S. schmuckeri, and
Archives of Insect Biochemistry and Physiology
246
Archives of Insect Biochemistry and Physiology, December 2009
S. sendtneri were assessed for the presence or absence of ecdysteroids commonly
associated with members of the genus Silene.
Comparison of the ecdysteroid profiles of the 7 ecdysteroid-positive samples
(Figs. 3 and 5) reveals how different they are since they can vary from quite simple
(e.g., S. mellifera) to highly complex (e.g., S. frivaldszkyana). Close analysis of the
chromatograms presented in Figure 5 clearly demonstrates the need for the use of
several HPLC systems involving different stationary and mobile phases, since one
system alone is not adequate to identify the ecdysteroids present with any certainty, not
only because non-ecdysteroidal compounds may elute at the retention time of
ecdysteroids, but also because certain ecdysteroid analogues co-chromatograph in
particular systems (e.g., 5b-H and 5b-OH analogues on C18-phases eluted with MeOH/
water mixtures).
As a first step in assessing the chemotaxonomic worth of ecdysteroid profiles, it
seemed reasonable to compare the ecdysteroid compositions of species within and
between Sections of the genus Silene. There have been several treatments of the
Sectional classification of Silene. Unfortunately, there is not full agreement between the
authors as to the number of Sections or the allocation of species to particular Sections
within this large genus, or even whether Silene forms a complex with species
traditionally allocated to other genera (e.g., Elisanthe, Lychnis, or Melandrium). The
allocation of the 7 species studied here to Sections according to the various authors is
summarised in Table 3. Chowdhuri (1957) recognised 443 species in the genus
Silene and allocated them to 44 Sections. Chater et al. (1993) recognise 194 European
species and allocate them to 29 Sections. Greuter (1995) recognises approximately 700
species and allocates them to 39 Sections. This considerably complicates the assessment
of phytoecdysteroids as chemotaxonomic markers at the Sectional level, such that
a priori the findings of this and previous studies on ecdysteroid profiles in Silene
extracts could only provide an initial assessment of the potential of this approach.
In conclusion, ecdysteroid profiling of species in the genus Silene is an effective way
of identifying species that contain high levels of ecdysteroids and new analogues. It is
still possible to identify many new ecdysteroid analogues even in extensively studied
genera such as Silene. Ecdysteroid profiles appear to be related to the Sectional
structure of the genus, but it is too early make definitive statements on this point,
owing to competing hypotheses concerning the Sectional structure (Chowdhuri, 1957;
Chater et al., 1993; Greuter, 1995; Zibareva et al., 2003a). The study of further key
Table 3. Allocation of the Studied Silene Species to Sections of the Genus According to Various
Authors (Chowdhuri, 1957; Chater et al., 1993; Greuter, 1995; Zibareva et al., 2003a)
Section
Species
S.
S.
S.
S.
S.
S.
S.
frivaldskyana
gigantean
graminifolia
mellifera
repens
schmuckeri
sendtneri
Chowdhuri
Flora Europaea
Greuter
Lazkov
Chloranthae
Paniculatae
–
Siphonomorpha
Spergulifoliae
Macranthae
Otites
Sclerocalycinae
Siphonomorpha
Graminifoliae
Siphonomorpha
Spergulifoliae
Spergulifoliae
Otites
Chloranthae
Italicae
–
–
–
–
Otites
Chloranthae
Siphonomorpha
Graminiformes
Siphonomorpha
Spergulifoliae
Saxifragoidea
Capitallatae
-, Species not recognised/treated.
Archives of Insect Biochemistry and Physiology
Phytoecdysteroid Profiles of 7 Species of Silene
247
species will clarify this relationship and may even help to resolve the debate about the
Sectional structure.
ACKNOWLEDGMENTS
We gratefully acknowledge financial support from CNRS and UPMC. L.Z. is grateful
for the opportunity of a research visit to UPMC.
LITERATURE CITED
Chater AO, Walters SM, Akeroyd JR, Wrigley F. 1993. Silene L. Flora Europaea, 2nd ed., vol. 1.
Cambridge, Cambridge University Press, p 191–218.
Chowdhuri PK. 1957. Studies in the genus Silene. Notes Roy Bot Garden Edinburgh
22:221–278.
Dinan L. 1989. Ecdysteroid structure and hormonal activity. In: Koolman J, editor. Ecdysone:
from chemistry to mode of action. Stuttgart: Georg Thieme Verlag. p 345–354.
Dinan L. 1995. Distribution and levels of phytoecdysteroids within individual plants of the
species of Chenopodiaceae. Eur J Entomol 92:295–300.
Dinan L. 2001. Phytoecdysteroids: biological aspects. Phytochemistry 57:325–339.
Dinan L. 2003. Ecdysteroid structure-activity relationships. In: Atta-ur-Rahman, editor. Natural
products chemistry, Vol. 29: bioactive natural products (Part J). Amsterdam: Elsevier
Scientific Publishing. p 3–71.
Dinan L, Lafont R. 2002. Compilation of the literature reports for the screening of vascular
plants, algae, fungi and non-arthropod invertebrates for the presence of ecdysteroids:
Version 2. Ecdybase, a free ecdysteroid database. http://ecdybase.org.
Dinan L, Lafont R. 2006. Effects and applications of arthropod steroid hormones (ecdysteroids)
in mammals. J Endocrinol 191:1–8.
Dinan L, Whiting P, Scott A. 1998. Taxonomic distribution of phytoecdysteroids in seeds of
members of the Chenopodiaceae. Biochem System Ecol 26:553–576.
Dinan L, Savchenko T, Whiting P. 2001. On the distribution of phytoecdysteroids in plants.
CLMS 58:1121–1132.
Girault JP. 1998. Determination of ecdysteroid structure by 1D and 2D NMR. Russian J Plant
Physiol 45:306–309.
Girault JP, Lafont R. 1988. The complete 1H-NMR assignment of ecdysone and
20-hydroxyecdysone. J Insect Physiol 34:701–706.
Greuter W. 1995. Silene (Caryophyllaceae) in Greece: a subgeneric and sectional classification.
Taxon 44:543–581.
Ho R, Girault J-P, Cousteau P-Y, Bianchini J-P, Raharivelomanana P, Lafont R. 2008. Isolation of
a new class of ecdysteroid conjugates (glucosyl-ferulates) using a combination of liquid
chromatographic methods. J Chromatogr Sci 46:102–110.
Lafont R, Harmatha J, Marion-Poll F, Dinan L, Wilson ID. 2002. Ecdybase, a free ecdysteroid
database. http://ecdybase.org.
Maria A, Girault JP, Saatov Z, Harmatha J, Dinan LN, Lafont R. 2005. Ecdysteroid glycosides:
chromatographic properties and biological activity. J Chrom Sci 43:149–157.
Nishimoto N, Shiobara Y, Inoue S-S, Fujino M, Takemoto T, Yeoh CL, de Oliveira F, Akisue G,
Akisue MK, Hashimoto G. 1988. Three ecdysteroid glycosides from Pfaffia iresinoides.
Phytochem 27:1665–1668.
Oxelman B, Lidén M, Berglund D. 1997. Chloroplast rps16 intron phylogeny of the tribe
Sileneae (Caryophyllaceae). Plant Syst Evol 206:393–410.
Archives of Insect Biochemistry and Physiology
248
Archives of Insect Biochemistry and Physiology, December 2009
Oxelman B, Lidén M, Rabeler RK, Popp M. 2001. A revised generic classification of the tribe
Sileneae (Caryophyllaceae). Nordic J Bot 20:743–748.
Revina TA, Revushkin AS, Rakitin AV. 1988. Ecdysteroid-containing species in flora of the Altai
mountains. Rastit Resur 34:565–570 (in Russian).
Saatov Z, Gorovits MB, Abubakirov NK. 1993. Phytoecdysteroids in plants of the genus Silene.
Khim Prirod Soedin 5:627–635 (in Russian).
Saatov Z, Agzamkhodzhaeva DA, Syrov VN. 1999. Distribution of phytoecdysteroids in plants of
Uzbekistan and the possiblity of using drugs based on them in neurological practice. Chem
Nat Comp 35:186–191.
Tomás J, Camps F, Claveria E, Coll J, Melé E, Messeguer J. 1992. Composition and location of
phytoecdysteroids in Ajuga reptans in vivo and in vitro cultures. Phytochem 31:1585–1591.
Zibareva LN. 1997. The prognosis of the presence of ecdysteroids among the species Silene L.
and Chenopodium L. to their contents in the seeds. Rastit Resur 33:89–92 (in Russian, with an
English abstract).
Zibareva L. 2000. Distribution and levels of phytoecdysteroids in plants of genus Silene during
development. Arch Insect Biochem Physiol 43:1–8.
Zibareva LN, Yeryomina VI. 1996. Dynamics of the contents of ecdysteroids in species of the
genus Silene L. grown in the Siberian botanical garden (Tomsk city). Rastit Resur
32:106–110 (in Russian, with an English abstract).
Zibareva L, Yeriomina V, Ivanova N, Lazkov G. 2003a. Distribution of phytoecdysteroids in
Sileneae Dumort. (Caryophyllaceae Juss.). Rastit Resur 39:45–53.
Zibareva L, Volodin V, Saatov Z, Savchenko T, Whiting P, Lafont R, Dinan L. 2003b. Distribution
of phytoecdysteroids in the Caryophyllaceae. Phytochem 64:499–517.
Zibareva LN, Dinan L, Eryomina VI. 2007. Screening of Caryophyllaceae species for
phytoecdysteroid presence. Rastitelny Resursy 43:66–75.
Archives of Insect Biochemistry and Physiology