ARCH 1108e Archives of Insect Biochemistry and Physiology 41:1–8 (1999)
Complex Phytoecdysteroid Cocktail of Silene otites (Caryophyllaceae) M. Báthori,1 J.-P. Girault,2 H. Kalasz,3 I. Mathé,1 L. N. Dinan,4 and R. Lafont5* 1
Dept Pharmacognosy, Albert Szent-Györgyi Medical University, Szeged, Hungary 2 Université René Descartes, CNRS URA 400, Paris, France 3 Dept Pharmacology, Semmelweis University of Medicine, Budapest, Hungary 4 Washington Singer Labs, University of Exeter, Exeter, UK 5 Ecole Normale Supérieure, Lab. Biochimie, CNRS EP 119, Paris, France Several new minor ecdysteroids from Silene otites (Caryophyllaceae) have been purified and identified. This plant species had previously been shown to contain a complex ecdysteroid cocktail, with 20-hydroxyecdysone as the major component, and significant amounts of 2-deoxyecdysone, 2-deoxy-20-hydroxyecdysone, and 20-hydroxyecdysone 22-acetate, and a set of minor ecdysteroids. The use of powerful techniques for purification and spectroscopic analyses has now allowed the isolation and identification of more than 30 different molecules including 21-hydroxylated ecdysteroids, thus adding a new position of the carbon skeleton that can be modified in ecdysteroids. Thus, in S. otites, a complex array of individual reactions can be used in various combinations leading both to major and minor components. Deciding whether this represents a random process without biological consequences or if (some of) the various minor components may play a specific role in, e.g., insect-plant relationships, will require the extensive use of appropriate in vitro and in vivo bioassays. Arch. Insect Biochem. Physiol. 41:1–8, 1999. © 1999 Wiley-Liss, Inc.
Key words: bioassay; Caryophyllaceae; HPLC; MS; NMR; phytoecdysteroid
Abbreviations used: amu = atomic mass unit; CI/D = chemical ionization/desorption; CIW = cyclohexane-isopropanol-water; COSY = correlation spectroscopy; DIW = dichloromethane-isopropanol-water; HPLC = high-performance liquid chromatography; MS = mass spectrometry; NMR = nuclear magrnetic resonance; NP = normal phase; PFG-HMBC = pulse field gradient spectroscopy–heteronuclear multiple-bond correlation; PFG-HMQC = pulse field gradient spectroscopy–heteronuclear multiple-quantum correlation; RP = reverse-phase; TLC = thin-layer chromatography; TOCSY = total correlation spectroscopY; 20E = 20-hydroxyecdysone; 2d20E = 2-deoxy-20hydroxyecdysone.
© 1999 Wiley-Liss, Inc.
Contract grant sponsor: Biotechnology and Biological Sciences Research Council. *Correspondence to: René Lafont, E.N.S., Laboratoire de Biochimie, CNRS EP 119, 46 rue d’Ulm, 75230 Paris Cedex 05, France. E-mail:
[email protected] Received 3 July 1998; accepted 27 December 1998
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INTRODUCTION Many plant species have been shown to contain large amounts of ecdysteroids, among which 20-hydroxyecdysone (20E), i.e., the major arthropod moulting hormone, is usually the most abundant one (Akhrem and Kovganko, 1989; Bergamasco and Horn, 1983; Camps, 1991; Kholodova, 1987; Lafont and Horn, 1989; Lafont et al., 1991; Lafont, 1998). When ecdysteroids are present, the plant usually contains a more or less complex cocktail of structurally different compounds, including major and minor ones. Among the plant kingdom, the Caryophyllaceae family has been shown to comprise many ecdysteroid-contaning species, for example in the Silene genus (see reviews by Báthori et al., 1987, and Saatov et al., 1993). We began a detailed investigation of Silene otites more than 10 years ago, and this plant species has already been the source of several new ecdysteroids (Báthori, 1986; Báthori et al., 1986a– c, 1988a,b, 1997; Girault et al., 1990, 1996). The availability of increasingly powerful analytical techniques presently allows the unambiguous identification of new ecdysteroids isolated in amounts of 0.1 mg or even less, and show that this plant species contains an extremely complex array of ecdysteroids. We report here the isolation and identification of several new ecdysteroids from a medium-scale extraction experiment. All these compounds have been tested using an in vitro bioassay, which shows that the different ecdysteroids display widely differing biological activities. MATERIALS AND METHODS Dried S. otites samples (aerial parts) were extracted and processed as usual (see Báthori et al., 1997; Girault et al., 1990). Two sequential preparative column chromatographies (the first on alumina, the second one on silica) allowed the separation of major and minor fractions, and further analysis of the minor fractions was performed using preparative TLC and/or HPLC. In the present experiments, preparative TLC was performed on silica plates (Merck, Darmstadt, Germany) developed with ethyl acetate–methanol– ammonia (85:10:5 v/v/v). The band of interest was scraped off and eluted with methanol. RP-TLC used
Whatman (Maidstone, Kent, UK) KC18F plates and 0.1% trifluoroacetic acid (TFA) in water-acetonitrile (65:35, v/v). NP-HPLC was performed on a semi-preparative column (Zorbax-SIL, 25 cm long, 9.4 mm i.d.) eluted with cyclohexane-isopropanol-water (CIW 100:40:3) as the mobile phase, and detection was performed at 245 nm. Other NP-HPLC solvent systems were also used for preparative and/or analytical purposes, i.e., dichloromethane-isopropanol-water (DIW 125:40:3 or 125:25:2). Mass spectrometry was performed on a Riber 10-10B (Nermag SA, Rueil-Malmaison, France) using a chemical ionization/desorption mode with ammonia as the reagent gas (Girault et al., 1990). Nuclear magnetic resonance (NMR) spectra were obtained as previously described (Girault, 1998; Girault and Lafont, 1988; Girault et al., 1990). Most of the ecdysteroids so far identified in S. otites were assayed for determining their activity in the BII cell assay system, which relies on a turbidimetric measurement of the inhibition of cell proliferation induced by ecdysteroids (Clément et al., 1993; Harmatha and Dinan, 1997). RESULTS S. otites contains a wide array of major and minor ecdysteroids, as evidenced from the analysis of a crude methanolic extract of the plant by NP-HPLC without any purification (Fig. 1). The content of 20E is approximately 0.75% relative to the dry weight of the plant. In the present study (Fig. 2), one minor fraction from the silica column chromatography step was further purified by preparative TLC and a band with a Rf = 0.29 (SO3) was scraped and eluted, then further analyzed by RP-TLC, giving three closely eluting components (A, B, C). The latter were further resolved using a NP-HPLC system, that gave an almost baseline separation of 6 compounds (Fig. 3A–C and Table 1). Those compounds were further analyzed by mass spectrometry (CI/D) and NMR. Four of them have been fully identified, and we report here the structures of compounds SO3-3 and SO3-4. Compounds SO3-5 and SO3-6 have been identified as dihydrorubrosterone and 5α-dihydrorubrosterone, respectively, and their full identification will be described elsewhere (Báthori et al., unpublished data).
Complex Phytoecdysteroid Cocktail of S. otites
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Fig. 1. NP-HPLC analysis of a crude alcoholic extract of Silene otites. Solvent system: dichloromethane-isopropanolwater (125:25:2), semi-preparative column (9.4 mm i.d.), flow-rate 4 ml.min–1.
CI/D mass spectrometry indicated a molecular mass of 464 for both SO3-3 and SO3-4, as for 2-deoxy-20-hydroxyecdysone (2d20E). A striking pecularity was the presence of a set of ions shifted by 30 amu corresponding to a loss of CH2O, indicative of the presence of one hydroxylated methyl group in the molecule. 1H NMR analysis (Table 2) showed that compound SO3-3 belongs to the 2 deoxy 5β-ecdysteroids as it presented no 2-H signal in the >CHOH area, a broadening of the 3-H signal (Girault et al., 1990), and the other
Fig. 2. General procedure for the isolation of ecdysteroids from Silene otites.
signals of the A- and B-rings were equivalent to those of 2d20E. However, further examination of the spectrum showed (1) the presence of only four methyl signals in the saturated region, (2) two new protons signals, δ = 3.90 ppm (d,d:11.2, 4.1) and δ = 3.76 ppm (d,d:11.2, 7.2) in the CHOH area. 2D experiments [COSY (Girault and Lafont, 1988), TOCSY (Bax and Davis, 1985), PFGHMQC and PFG-HMBC (Hurd and John, 1991)]
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Fig. 3. NP-HPLC analysis (solvent: CIW 100:40:3, semipreparative column) of the three SO3 fractions (A–C) from
RP-TLC. The numbers refer to compounds SO3-1 to SO3-6 from Figure 2.
showed that these two new proton signals could be assigned as the diastereotopic protons of the hydroxylated C-21. Indeed, the 2D COSY experiment showed that they were coupled together and with a common partner H-20, which was corre-
lated to H-17 (these proton signals being assigned by 2D COSY, TOCSY, PFG-HMQC, and PFGHMBC analysis). This assignment was confirmed based on 2D PFG-HMQC, which showed that these two protons were bound to the same car-
TABLE 1. The 6 Ecdysteroid Components From the SO3 Fraction (HPLC used CIW 100:40:3 as Solvent System, Flow-Rate 4 mL.min–1) Peak number
Retention time (min)
SO3-1
11.6
SO3-2
12.6
SO3-3
14.8
SO3-4
17.2
SO3-5 SO3-6
20.2 22.9
Major ions (CI/D) 482 (M+H+NH3)+, 465 (M+H), 447, 429, 411, 393, 364, 347, 329, 320, 303, 285, 162, 144, 127 (base peak) 482 (M+H+NH3)+, 465 (M+H), 447 (base peak), 429, 411, 393, 364, 347, 329, 320, 303, 285, 160, 143, 116 482 (M+H+NH3)+, 465 (M+H), 452, 447 (base peak), 435, 429, 417, 411, 399, 393, 366, 349, 331, 313, 116 482 (M+H+NH3)+, 465, (M+H), 452, 447, 435, 429 (base peak), 417, 411 399, 393, 366, 349, 331, 313, 116 354 (M+H+NH3)+, 337 (M+H) (base peak), 319, 301 354 (M+H+NH3)+, 337 (M+H) (base peak), 319, 301
Molecular mass 464 464 464 464 336 336
Complex Phytoecdysteroid Cocktail of S. otites
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TABLE 2. Chemical Shifts (1H) of SO3-3, SO3-4 and 2d20E in D2O* Proton number
SO3-3
SO3-4
2d20E
1-Hax 1-Heq 2-Hax 2-Heq 3-Heq 4-Hax 4-Heq 5-H 7-H 9-Hax 11-Hax 11-Heq 12-Hax 12-Heq 15-Hax 15-Heq 16-Hax 16-Heq 17-H 18-Me 19-Me 20-H 21-Me 21-Ha 21-Hb 22-Hb 23-Ha 23-Hb 24-Ha 24-Hb 26-Me 27-Me
1.36 1.85 1.36a 1.68a 4.10 (m, w½ = 25) 1.62 1.76 2.40 (d,d, 12.5, 2) 5.97 (d, 2) 3.14 (m, w½ = 26) 1.66 1.81 1.91 1.75 2.09 1.64 1.97 1.62 2.07 0.77 (s) 0.98 (s) 1.92 — 3.90 (d,d, 11.2, 4.1) 3.76 (d,d, 11.2, 7.2) 3.78 1.53 1.67 1.48 1.82 1.236 (s) 1.243 (s)
1.49 1.66 1.49a 1.81a 3.70 (m, w½ = 29) 1.39 2.09 2.58 (d,d 12.1, 2) 5.99 (d, 2) 2.74 (m, w½ = 25) 1.64 1.81 1.93 1.73 2.05 1.65 1.95 1.63 2.05 0.77 (s) 0.87 (s) 1.94 — 3.91 (d,d, 11.4, 4) 3.77 (d,d, 11.4, 7) 3.79 1.53 1.67 1.49 1.83 1.236 (s) 1.243 (s)
1.38 1.85 1.38a 1.65a 4.11 (m, w½ = 23) 1.62 1.75 2.40 (d,d, 12, 2) 5.97 (d, 1.8) 3.16 (m, w½ = 26) 1.71 1.84 1.96 1.70 2.07 (m) 1.65 1.90 1.80 2.34 (t, 9.3) 0.87 (s) 0.98 (s) — 1.24 (s) — — 3.43 (d, 10) 1.33 1.65 1.51 (d, t 12.5, 3.6) 1.80 1.23 (s) 1.24 (s)
*ax: axial; eq: equatorial; a: signals can be interverted; s: singlet; d: doublet; t: triplet; w½: width at half-height.
bon (δ = 62.2 ppm) and from 2D PFG-HMBC longrange 1H-13C correlations. One observed the correlations of these two protons with C-20, C-22, and C-17 and also from H-22 to C-24 and to the new CH2OH carbon (Hurd and John, 1991). 1 H and 13C NMR analysis (Tables 2 and 3) showed that compound SO3-4 presented the same structure of the side-chain (see above), and was a 2-deoxyecdysteroid because of the lack of a 2-H signal in the >CHOH area and a broadening of the 3H signal (Girault et al., 1990), but with a 5α configuration for the junction of rings A and B. This was established from the signals of the A- and Brings: a downfield shift of H-5 and an upfield shift of H-9, H-3, and 19-methyl (Girault et al., 1990). This was confirmed thanks to a large 13C NMR chemical shift of C-19 (δ = 13.1 ppm) for SO3-4 with respect to (δ = 24.2 ppm) for SO3-3. An inversion of
ring A causes significant shifts of the carbons signals of rings A and B. In 5β-steroids, the C-19 methyl carbon resonates about 12 ppm towards lower field than in the corresponding 5α-steroid (Breitmeier and Voelter, 1987). So, the structures of SO33 and SO3-4 (M = 464) were definitely assigned as 2-deoxy-21-hydroxyecdysone and 5α-2-deoxy-21hydroxyecdysone, respectively (Fig. 4). Bioassays showed that these two new ecdysteroids have a low biological activity (ED50 = 4.3 × 10–6 M and 9.5 × 10–5 M, respectively, for SO3-3 and SO3-4). These values are far higher than that of 20E (7.5 × 10–9 M). The activity of SO3-3 is intermediate between that of 2dE and 2d20E, and the 5α-isomer is less active, but it still displays a noticeable biological activity. The ED50 values were also determined for most of the other ecdysteroids isolated from S. otites and are reported in Table 4.
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TABLE 3. Chemical Shifts (13C) of SO3-3, SO3-4, and 2d20E in D2O Carbon no. C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-20 C-21 C-22 C-23 C-24 C-25 C-26 C-27
Multiplicity
SO3-3
SO3-4
CH2 CH2 CH-OH CH2 CH C=O =CH =C CH C CH2 CH2 C C-OH CH2 CH2 CH CH3 CH3 CH CH2-OH CH-OH CH2 CH2 C-OH CH3 CH3
29.2
37.1
28.6 28.0 65.7
52.3
54.2
52.3
121.8
123.4
121.8
37.4 37.4 21.0 31.2 48.0 86.5 31.4 26.4 44.4 16.6 24.2 48.5 62.2 75.8 27.1 41.8 72.7 28.6 29.2
46.9 39.6
37.1 37.1 21.0 31.9 48.3 86.1 31.1 21.0 50.3 18.0 24.1 78.9 20.6 (CH3) 78.3 27.0 41.6 72.5 28.3 29.2
65.7
30.8 47.4 86.1
44.4 16.5 13.1 48.5 62.0 75.3 27.1 41.6 72.7 28.6 29.2
2d20E
DISCUSSION About Ecdysteroid Diversity: How Many Reactions Generate This Diversity? When considering the ecdysteroids listed in Table 4, we observe that a large set of individual changes may occur. By reference to 20E, we can notice: (1) ± hydroxylation at positions 1, 2, 20, 21, and 26; (2) esterification with acetate at positions
Fig. 4.
2, 3, 22, 25, with crotonate at position 3, with benzoate at position 22; (3) etherification with glucose at positions 3 and 25; (4) side-chain cleavage giving rise to 24C, 21C, or 19C ecdysteroids; (5) isomerization at position 5. The list is not complete. Considering a combination of all these individual changes, we can expect more than 105 different ecdysteroids to be found. Of course, some of these reactions cannot take place at the same time (i.e., two conjugations at the same position, or conjugation at position 25 and side-chain cleavage), but still a very large number of ecdysteroids should theoretically be formed. So most probably the number of the isolated ecdysteroids does not reflect the diversity of the compounds present. The rationale of all these modifications is also unclear: while some changes lead to less polar derivatives (acetates, diacetates, …), other ones increase water solubility (glucosides). If the second case appears logical for compounds to be stored in cell vacuoles, the former has not yet received any explanation. It would be of great interest to obtain more information about the subcellular localization of the various ecdysteroids. Biological Activity: Structure-Activity Relationships Studies The BII bioassay measures in an accurate way the affinity of ecdysteroids to the ecdysone receptor (Clément et al., 1993; Harmatha and Dinan, 1997). Its use with many different ecdysteroids is aimed to determine the structural features of ecdysteroids associated with a high
Structures of the two new ecdysteroids described in the present study.
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TABLE 4. Biological Activity (ED50) of the Different Ecdysteroids of S. otites Using the BII Cell Assay Type of ecdysteroids 27C, free ± OH group(s)
Simple conjugates Acetates
Benzoates Crotonates Glucosides Double conjugates Homoconjugates Heteroconjugates 28-C ecdysteroids Side-chain cleavage products 24C 21C 19C
Name
ED50 × × × × × × × × ×
10–9 M 10–5 M 10–7 M 10–7 M 10–6 M 10–6 M 10–6 M 10–6 M 10–5 M
20-hydroxyecdysone (20E) 2dE 2d20E Integristerone A 2-Deoxyintegristerone A Ecdysone 20, 26E 2d21E 5α-2d21E
7.5 5.0 6.6 1.8 7.0 1.1 7.3 4.3 9.5
2dE 22-acetate 2d20E 22-acetate 20E 22-acetate 20E 2-acetate 20E3-acetate 20E 25-acetate 2d20E 3-acetate 5α-2d20E 3-acetate 2d20E22-benzoate 2d20E 3-crotonate 20E 25-glucoside 20E 3-glucoside
1.4 × 10–6 M 2.4 × 10–5 M 4.0 × 10–6 M 4.0 × 10–7 M 4.7 × 10–7 M 1.0 × 10–7 M 4.3 × 10–6 M Not tested 6.3 × 10–6 M Not tested 8.5 × 10–6 M 1.3 × 10–5 M
2d20E 3,22-diacetate 20E 22-benzoate-25-glucoside Makisterone A 24,28-Dehydromakisterone A
Inactive Not tested 1.3 × 10–8 M 4.0 × 10–9 M
Sidisterone Poststerone Dihydropoststerone Rubrosterone Dihydrorubrosterone 5α-Dihydrorubrosterone
> 10–4 M 2 × 10–5 M 7.5 × 10–4 M > 10–4 M Inactive 5.6 × 10–6 M
biological activity. In this bioassay, the most active ecdysteroid is ponasterone A (ED50 = 3.1 × 10–10 M) followed by 20E and related 28-C ecdysteroids. The bioassays show that the different ecdysteroids from S. otites are all less active than 20-hydroxyecdysone. When a single position is modified, the activity is usually lower by at least one order of magnitude, and when more profound changes are present, the molecules can become inactive (i.e. ED50 > 10–4 M). This, however, does not exclude the possibility that other kinds of bioassays could provide different results, especially in vivo bioassays where a larger set of parameters are involved in the biological activity such as uptake, detoxification mechanisms, and metabolism (see discussion in Blackford, 1995). Ecdysteroids may also display deterrency activities towards insects (e.g., Tanaka et al., 1994), and they could be important in insect-plant relationships. Similarly, deterrent
activities of ecdysteroids have been demonstrated in the crab Carcinus maenas, and the structureactivity relationships in this case are totally different from those involving the classical nuclear receptors (Guckler et al., 1998). ACKNOWLEDGMENTS L.D. thanks Pensri Whiting for excellent technical support, and the Biotechnology and Biological Sciences Research Council for funding. LITERATURE CITED Akhrem AA, Kovganko NV. 1989. Ecdysteroids: chemistry and biological activity. Minsk: Nauka i Technika. 327 pp. Báthori M. 1986. HPLC analysis of ecdysteroids of Silene otites (L): Wib. In: Kalasz H, Ettre LS, editors. Chromatography ’84, Budapest: Akademiai Kiado. p 297–306. Báthori M, Szendrei K, Herke I. 1986a. The ecdysteroids of Silene otites (L):Wib. Herba Hung 25:105–117.
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Lafont R. 1998. Phytoecdysteroids in the world flora: diversity, distribution, biosynthesis and evolution. Russ J Plant Physiol 45:276–295. Lafont R, Horn DHS. 1989. Phytoecdysteroids: structures and occurrence. In: Koolman J, editor. Ecdysone. From chemistry to mode of action. Stuttgart: Georg Thieme Verlag. p 39–64. Lafont R, Bouthier A, Wilson ID. 1991. Phytoecdysteroids: structures, occurrence, biosynthesis and possible ecological significance. In: Hrdy I, editor. Insect chemical ecology. The Hague: Academia Praha and SBP Acad Pub. p 197–214. Saatov Z, Gorovits MB, Abubakirov NK. 1993. Phytoecdysteroids from the Silene genus. Khim Prir Soedin 627–635. Tanaka Y, Asaoka K, Takeda S. 1994. Different feeding and gustatory responses to ecdysone and 20-hydroxyecdysone by larvae of the silkworm, Bombyx mori. J Chem Ecol 20:125–133.
