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. 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