The complex metabolism of poststerone in male rats.

Ecdysteroids are not endogenous to mammals, but are normal components of the food intake of many mammalian species consuming phytoecdysteroid-containing plants. The most frequently encountered phytoecdysteroid is 20-hydroxyecdysone (20E). Several pharmaceutical effects have been observed after ecdysteroid injection or ingestion, but it is not clear to what extent metabolites generated in the mammalian body contribute to these effects. The C21-ecdysteroid poststerone (Post) is a metabolite of 20E in rodents. Post analogues are key intermediates in the metabolism of exogenous ecdysteroids possessing a C20/22-diol. The pharmacokinetics, bioavailability and metabolism of Post have been assessed in male rats after ingestion and injection. The bioavailability of Post is significantly greater than that of 20E and the presence of an efficient entero-hepatic cycle allows Post to be effectively metabolised to a wide range of metabolites which are excreted mainly in the faeces, but also to some extent in the urine. Several of the major metabolites in the bile have been identified unambiguously as 3-epi-poststerone, 16α-hydroxypoststerone, 21-hydroxypoststerone and 3-epi-21-hydroxypoststerone. Conjugates are also present. Parallels are drawn to the metabolism of endogenous vertebrate steroid hormones, to which Post bears more similarity than 20E.

The minor ecdysteroids from Ajuga turkestanica.

INTRODUCTION: Ajuga turkestanica is a plant used in traditional medicine for its high ecdysteroid content, including the presence of the particularly active turkesterone, which possess efficient anabolic activity. OBJECTIVES: To isolate and identify minor ecdysteroids present in a semi-purified plant fraction containing ca. 70% turkesterone. MATERIAL AND METHODS: Multi-step preparative HPLC (combining RP- and NP-HPLC systems) was used to purify the different components present in the turkesterone fraction. Isolated compounds were identified by high-resolution mass spectrometry and 2D-NMR. RESULTS: Fourteen ecdysteroids (including turkesterone and 20-hydroxyecdysone) were isolated. Seven of these, all bearing an 11α-hydroxy group, were previously unreported. CONCLUSION: Ajuga turkestanica ecdysteroids are characterised by the abundance of 11α-hydroxylated compounds and by the simultaneous presence of 24C, 27C, 28C and 29C ecdysteroids. It is expected that even more ecdysteroids are to be found in this plant since the starting material for this study lacked the less polar ecdysteroids. The simultaneous presence of 20-hydroxyecdysone and turkesterone (its 11α-hydroxy analogue) as the two major ecdysteroids suggests that every ecdysteroid is probably present in both 11α-hydroxy and 11-deoxy forms.

Ecdysteroid profiles of two Ajuga species, A. iva and A. remota.

Phytoecdysteroids are plant analogues of insect moulting hormones and are used by plants to repel or disturb phytophagous insects. They are also active on mammals and present in many plants used in traditional medicine. The Ajuga genus contains several such species, which occur in various pharmacopoeias. We report the isolation and identification of major and minor ecdysteroids present in two Ajuga species, A. iva and A. remota, both of which are used as medicinal plants in Africa. Three minor ecdysteroids (abutasterone, ponasterone A and sidisterone) have been found for the first time in the Ajuga genus.

Stereochemical assignment of C-24 and C-25 of amarasterone A, a putative biosynthetic intermediate of cyasterone.

A C29 phytoecdysteroid named amarasterone A (1) has been isolated from Cyathula capitata (Amaranthaceae), Leuzea carthamoides (Asteraceae), and Microsorum scolopendria (Polypodiaceae). We recently isolated amarasterone A from C. officinalis. Amarasterone A has been postulated as a biosynthetic intermediate of cyasterone in Cyathula sp. The stereochemistry at the C-24 and C-25 positions of these amarasterone A samples was investigated by comparing the NMR spectroscopic data with those of stereodefined model compounds, (24R,25S)-, (24R,25R)-, (24S,25S)-, and (24S,25R)-isomers of (20R,22R)-3ß-methoxystigmast-5-ene-20,22,26-triol (2a-d), which were synthesized in the present study. Amarasterone A isolated from Cyathula officinalis was determined to be the (24R,25S)-isomer (1a), while amarasterone A from L. carthamoides was found to be the (24R,25R)-isomer (1b). Amarasterone A from M. scolopendria was found to be a mixture of 1a and 1b. The biosynthesis of cyasterone in Cyathula sp. is discussed on the basis of the identical C-24 configuration of sitosterol and amarasterone A.

E- and Z-isomers of new phytoecdysteroid conjugates from French Polynesian Microsorum membranifolium (Polypodiaceae) fronds.

Phytochemical investigation of the fronds of Microsorum membranifolium resulted in the isolation of a new phytoecdysteroid, E-2-deoxy-20-hydroxyecdysone 3-[4-(1-ß-D-glucopyranosyl)]-caffeate (1), together with two known phytoecdysteroids, E-2-deoxy-20-hydroxyecdysone 3-[4-(1-ß-D-glucopyranosyl)]-ferulate (2), E-2-deoxyecdysone 3-[4-(1-ß-D-glucopyranosyl)]-ferulate (3). Their respective Z-isomers 4-6 were also observed and identified for the first time. The new structures were elucidated on the basis of extensive spectroscopic data analysis (1D, 2D-NMR and HR-MS techniques).

Two minor phytoecdysteroids of the plant Silene viridiflora.

Chemical investigations of Silene viridiflora (L.) yielded a new ecdysteroid, 20-hydroxyecdysone 20,22-monoacetonide-25-acetate (1), and a known ecdysteroid, 2-deoxypolypodine B-3-beta-D-glucoside (2). The elucidation of the chemical structures was established by 1D and 2D NMR experiments.

Automatic clustering of docking poses in virtual screening process using self-organizing map.

MOTIVATION: Scoring functions provided by the docking software are still a major limiting factor in virtual screening (VS) process to classify compounds. Score analysis of the docking is not able to find out all active compounds. This is due to a bad estimation of the ligand binding energies. Making the assumption that active compounds should have specific contacts with their target to display activity, it would be possible to discriminate active compounds from inactive ones with careful analysis of interatomic contacts between the molecule and the target. However, compounds clustering is very tedious due to the large number of contacts extracted from the different conformations proposed by docking experiments. RESULTS: Structural analysis of docked structures is processed in three steps: (i) a Kohonen self-organizing map (SOM) training phase using drug-protein contact descriptors followed by (ii) an unsupervised cluster analysis and (iii) a Newick file generation for results visualization as a tree. The docking poses are then analysed and classified quickly and automatically by AuPosSOM (Automatic analysis of Poses using SOM). AuPosSOM can be integrated into strategies for VS currently employed. We demonstrate that it is possible to discriminate active compounds from inactive ones using only mean protein contacts' footprints calculation from the multiple conformations given by the docking software. Chemical structure of the compound and key binding residues information are not necessary to find out active molecules. Thus, contact-activity relationship can be employed as a new VS process. AVAILABILITY: AuPosSOM is available at http://www.aupossom.com.

Ecdysteroids from Cyanotis longifolia Benth. (Commelinaceae).

Cyanotis longifolia Benth. (Commelinaceae) contains ecdysteroids, which are highly concentrated in the roots and flowers, whereas leaves contain only very low amounts and stems intermediate amounts. 20-Hydroxyecdysone is the major component found in all tissues, but roots also contain large amounts of 20-hydroxyecdysone 3-acetate and ajugasterone C. A preparative experiment has shown that roots contain a complex ecdysteroid mixture, and the analysis of minor components has allowed the isolation of several already known ecdysteroids (polypodine B, 2-deoxy-20,26-dihydroxyecdysone, isovitexirone, poststerone) together with five new (ajugasterone C 3-acetate, 5beta-hydroxy-poststerone, poststerone 2-acetate, 14(15)-dehydro-poststerone 2-acetate, 24-epi-atrotosterone A [=24-methyl-ajugasterone C]) ecdysteroids that have been fully characterized. A preliminary investigation of 55 species belonging to 15 different genera of the Commelinaceae has shown that several of them contain significant concentrations of ecdysteroids, among which some previously uninvestigated ones appear to be very promising sources of ecdysteroids.

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

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-beta-D-glucoside, which is a new ecdysteroid. Ecdysteroids in the other 5 species have been identified by co-chromatography with reference 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 discussed.

Structure of the complex between phosphorylated substrates and the SCF beta-TrCP ubiquitin ligase receptor: a combined NMR, molecular modeling, and docking approach.

The binding of phosphorylated peptides to the receptor plays a major role in many basic cellular processes in a variety of pathological states. Human beta-TrCP is a key component of a recently characterized E3 ubiquitin ligase complex that regulates protein degradation through the ubiquitin-dependent proteasome pathway. Docking studies were carried out to explore the structural requirements for the beta-TrCP substrates. Docking studies were performed on the bound conformation of the phosphorylated peptides determined by NMR, whereas the beta-TrCP structure was derived by X-ray from Protein Data Bank. After the docking calculation, during which the peptides were conformationally restrained, the complex presented herein was analyzed in terms of ligand-protein interactions and properties of contacting surfaces. The structural requirements for phosphorylated substrates in interaction with beta-TrCP were explored and compared with experimental data from TRNOESY and STD NMR results. The analysis revealed that the bend of the peptide structures, which is indispensable for beta-TrCP recognition, aligns two charged phosphate groups and a central hydrophobic group in a favorable arrangement that leads to the burial of the peptide surface in the binding cleft upon complexation. Through docking simulations, we have identified different specific binding pockets of beta-TrCP according to the ligand in interaction. These data should be valuable in the rational design of a ligand to be used in therapeutic approaches.

The key-role of tyrosine 155 in the mechanism of prion transconformation as highlighted by a study of sheep mutant peptides.

Prion protein is a strongly conserved and ubiquitous glycoprotein. The conformational conversion of the non-pathogenic cellular prion isoform (PrP(C)) into a pathogenic scrapie isoform (PrP(Sc)) is a fundamental event in the onset of transmissible spongiform encephalopathies (TSE). During this conversion, helix H1 and its two flanking loops are known to undergo a conformational transition into a beta-like structure. In order to understand mechanisms which trigger this transconformation, sheep prion protein synthetic peptides spanning helix 1 and beta-strand 2 (residues 142-166) were studied: (1) the N3 peptide, studied earlier, is known to fold into beta-hairpin-like conformation in phosphate buffer at neutral pH and to adopt a helix H1 conformation when dissolved in trifluoroethanol/phosphate buffer mixture, (2) The R156A mutant (peptide R15) and (3) the Y155A mutant (peptide Y14) of the N3 peptide are studied by circular dichroism and NMR spectroscopy in this article. Structural characterization of these peptides highlights the key role of tyrosine 155 in the stabilization of the beta-hairpin-like conformation of the sheep peptide in phosphate buffer. We propose a model where tyrosine 155 could stabilize the beta-hairpin structure by creating a hydrophobic core in phosphate buffer, necessary to initiate the beta-type structure formation. In the turn, the side chain ionic interaction, E152-R156 described before, seems to play a minor role relative to the hydrophobic packing, as observed with the R156A mutation (peptide R15). Interestingly, homology at amino acid residue 155 could be responsible for the species barrier in TSE.

Isolation of a new class of ecdysteroid conjugates (glucosyl-ferulates) using a combination of liquid chromatographic methods.

The Polynesian medicinal fern Microsorum membranifolium contains very large amounts of ecdysteroids, including ecdysone, 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone, and 2-deoxyecdysone. It also contains large amounts of unusual ecdysteroids which have been unambiguously identified by mass spectrometry and nuclear magnetic resonance. A new class of ecdysteroid conjugates (3-glucosyl-ferulates of 2-deoxyecdysone and 2-deoxy-20-hydroxyecdysone) is isolated, together with a new glycoside (2-deoxyecdysone 25-rhamnoside). The simultaneous presence of a sugar and an aromatic moiety results in a very particular chromatographic behavior of these conjugates. They behave like flavonoids and polyphenols when using the classical purification on polyamide, aimed at removing the latter from crude plant extracts, and would therefore be lost. They elute as non-polar ecdysteroids on reversed-phase high-performance liquid chromatography (RP-HPLC), whereas their behavior on normal-phase (NP) HPLC is strongly dependent on the mobile phase composition. Our data highlight the importance of selectivity in the choice of HPLC methods used for ecdysteroid separations.

Transfer-NMR and docking studies identify the binding of the peptide derived from activating transcription factor 4 to protein ubiquitin ligase beta-TrCP. Competition STD-NMR with beta-catenin.

ATF4 plays a crucial role in the cellular response to stress. The E3 ubiquitin ligase, SCF beta-TrCP protein responsible for ATF4 degradation by the proteasome, binds to ATF4 through a DpSGXXXpS phosphorylation motif, which is similar but not identical to the DpSGXXpS motif found in most other substrates of beta-TrCP. NMR studies were performed on the free and bound forms of a peptide derived from this ATF4 motif that enabled the elucidation of the conformation of the ligand complexed to the beta-TrCP protein and its binding mode. Saturation transfer difference (STD) NMR allowed the study of competition for binding to beta-TrCP, between the phosphorylation motifs of ATF4 and beta-catenin, to characterize the ATF4 binding epitope. Docking protocols were performed using the crystal structure of the beta-catenin-beta-TrCP complex as a template and NMR results of the ATF4-beta-TrCP complex. In agreement with the STD results, in order to bind to beta-TrCP, the ATF4 DpSGIXXpSXE motif required the association of two negatively charged areas, in addition to the hydrophobic interaction in the beta-TrCP central channel. Docking studies showed that the ATF4 DpSGIXXpSXE motif fits the binding pocket of beta-TrCP through an S-turning conformation. The distance between the two phosphate groups is 17.8 A, which matched the corresponding distance 17.1 A for the other extended DpSGXXpS motif in the beta-TrCP receptor model. This study identifies the residues of the beta-TrCP receptor involved in ligand recognition. Using a new concept of STD competition experiment, we show that ATF4 competes and inhibits binding of beta-catenin to beta-TrCP.

Phosphorylation-dependent structure of ATF4 peptides derived from a human ATF4 protein, a member of the family of transcription factors.

ATF4 plays a crucial role in the cellular response to stress and the F-box protein beta-TrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for ATF4 degradation by the proteasome, binds to ATF4, and controls its stability. Association between the two proteins depends on ATF4 phosphorylation of serine residues 219 and 224 present in the context of DpSGXXXpS, which is similar but not identical to the DpSGXXpS motif found in most other substrates of beta-TrCP. We used NMR spectroscopy to analyze the structure of the 23P-ATF4 peptide. The 3D structure of the ligand was determined on the basis of NOESY restraints that provide an hairpin loop structure. In contrast, no ordered structure was observed in the NMR experiments for the nonphosphorylated 23-ATF4 in solution. This structural study provides information, which could be used to study the beta-TrCP receptor-ligand interaction in docking procedure. Docking studies showed that the binding epitope of the ligand, is represented by the DpSGIXXpSXE motif. 23P-ATF4 peptide fits the binding pocket of protein beta-TrCP very well, considering that the DpSGIXXpSXE motif adopts an S-turning conformation contrary to the extended DpSGXXpS motif in the other known beta-TrCP ligands.

Ecdysteroids from the medicinal fern Microsorum scolopendria (Burm. f.).

Fronds of the fern Microsorum scolopendria are widely used in traditional medicine in the Society Islands. They were investigated for the presence of ecdysteroids, which might be responsible for at least some of their medicinal properties. M. scolopendria represents an excellent source of ecdysone (0.16% of dry weight) and 20-hydroxyecdysone (0.20%), and also contains significant amounts (0.01-0.02%) of makisterones A and C, inokosterone and amarasterone A, together with lower amounts of poststerone and of a compound tentatively identified as 24,28-diepi-cyasterone. During this study, three new minor phytoecdysteroids, namely 20-deoxymakisterone A, a 25(?)-epimer of amarasterone A and 25-deoxyecdysone 22-glucoside were also isolated by a combination of normal- and reversed-phase HPLC and subsequently identified by NMR.

Structural studies on 24P-IkappaBalpha peptide derived from a human IkappaB-alpha protein related to the inhibition of the activity of the transcription factor NF-kappaB.

The IkappaB-alpha protein, inhibitor of the transcription factor nuclear factor-kappaB (NF-kappaB), is a cellular substrate of beta-transducin repeat containing protein (beta-TrCP). beta-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. beta-TrCP targets the protein IkappaB-alpha for ubiquitination, followed by proteasome degradation. The SCF-beta-TrCP complex specifically recognizes an IkappaB-alpha peptide containing the DpSGXXpS motif in a phosphorylation-dependent manner. A fragment comprising 24 amino acids residues for the phosphorylated peptide at the two sites Ser32 and Ser36 and thus termed 24P-IkappaBalpha (P-IkappaBalpha21-44) was characterized conformationally by NMR spectroscopy and molecular dynamics simulation. In the free states, 24P-IkappaBalpha exhibits mainly a random coil conformation, although the presence of a nascent bend was detected between residues 30 and 36, flanked by two N- and C-terminal disordered regions. The bound conformation of the phosphorylated IkappaB-alpha peptide was obtained using transfer nuclear Overhauser effect spectroscopy (TRNOESY) experiments. To further elucidate the basis of the beta-TrCP interaction, a complex between 24P-IkappaBalpha peptide and beta-TrCP protein was studied using saturation transfer difference (STD) NMR experiments. The conformation of 24P-IkappaBalpha bound to beta-TrCP presents a bend corresponding to the 31DpSGLDpS36 motif and on both sides N- and C-terminal turn regions (Lys22-Asp31 and Met37-Glu43). The bound structure of the phosphorylated peptide suggests that these domains are crucial for the interaction of the peptide with its receptor showing the protons identified by STD NMR as exposed in close proximity to the beta-TrCP surface.

STD and TRNOESY NMR studies for the epitope mapping of the phosphorylation motif of the oncogenic protein beta-catenin recognized by a selective monoclonal antibody.

The interaction of the P-beta-Cat(19-44) peptide, a 26 amino acid peptide (K(19)AAVSHWQQQSYLDpSGIHpSGATTTAP(44)) that mimics the phosphorylated beta-Catenin antigen, has been studied with its monoclonal antibody BC-22, by transferred nuclear Overhauser effect NMR spectroscopy (TRNOESY) and saturation transfer difference NMR (STD NMR) spectroscopy. This antibody is specific to diphosphorylated beta-Catenin and does not react with the non-phosphorylated protein. Phosphorylation of beta-Catenin at sites Ser33 and Ser37 on the DSGXXS motif is required for the interaction of beta-Catenin with the ubiquitin ligase SCF(beta-TrCP). beta-TrCP is involved in the ubiquitination and proteasome targeting of the oncogenic protein beta-Catenin, the accumulation of which has been implicated in various human cancers. The three-dimensional structure of the P-beta-Cat(19-44) in the bound conformation was determined by TRNOESY NMR experiments; the peptide adopts a compact structure in the presence of mAb with formation of turns around Trp25 and Gln26, with a tight bend created by the DpS(33)GIHpS(37) motif; the peptide residues (D32-pS37) forming this bend are recognized by the antibody as demonstrated by STD NMR experiments. STD NMR studies provide evidence for the existence of a conformational epitope containing tandem repeats of phosphoserine motifs. The peptide's epitope is predominantly located in the large bend and in the N-terminal segment, implicating bidentate association. These findings are in excellent agreement with a recently published NMR structure required for the interaction of beta-Catenin with the SCF(beta-TrCP) protein.

NMR studies for identifying phosphopeptide ligands of the HIV-1 protein Vpu binding to the F-box protein beta-TrCP.

The human immunodeficiency virus type 1 (HIV-1) Vpu enhances viral particle release and, its interaction with the ubiquitin ligase SCF-beta-TrCP triggers the HIV-1 receptor CD4 degradation by the proteasome. The interaction between beta-TrCP protein and ligands containing the phosphorylated DpSGXXpS motif plays a key role for the development of severe disease states, such as HIV or cancer. This study examines the binding and conformation of phosphopeptides (P1, LIERAEDpSG and P2, EDpSGNEpSE) from HIV protein Vpu to beta-TrCP with the objective of defining the minimum length of peptide needed for effective binding. The screening step can be analyzed by NMR spectroscopy, in particular, saturation transfer NMR methods clearly identify the residues in the peptide that make direct contact with beta-TrCP protein when bound. An analysis of saturation transfer difference (STD) spectra provided clear evidence that the two peptides efficiently bound beta-TrCP receptor protein. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated peptides was determined using transferred NOESY methods, which gave rise to a well-defined structure. P1 and P2 can fold in a bend arrangement for the DpSG motif, showing the protons identified by STD-NMR as exposed in close proximity at the molecule surface. Ser phosphorylation allows electrostatic interaction and hydrogen bond with the amino acids of the beta-TrCP binding pocket. The upstream LIER hydrophobic region was also essential in binding to a hydrophobic pocket of the beta-TrCP WD domain. These findings are in good agreement with a recently published X-ray structure of a shorter beta-Catenin fragment with the beta-TrCP complex.

STD and TRNOESY NMR studies on the conformation of the oncogenic protein beta-catenin containing the phosphorylated motif DpSGXXpS bound to the beta-TrCP protein.

beta-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. Biochemical studies have suggested that beta-TrCP targets the oncogenic protein beta-catenin for ubiquitination and followed by proteasome degradation. To further elucidate the basis of this interaction, a complex between a 32-residue peptide from beta-catenin containing the phosphorylated motif DpSGXXpS (P-beta-Cat17-48) and beta-TrCP was studied using Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) experiments. These experiments make it possible to identify the binding epitope of a ligand at atomic resolution. An analysis of STD spectra provided clear evidence that only a few of the 32 residues receive the largest saturation transfer. In particular, the amide protons of the residues in the phosphorylated motif appear to be in close contact to the amino acids of the beta-TrCP binding pocket. The amide and aromatic protons of the His24 and Trp25 residues also receive a significant saturation transfer. These findings are in keeping with a recently published x-ray structure of a shorter beta-catenin fragment with the beta-TrCP1-Skp1 complex and with the earlier findings from mutagenesis and activity assays. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated beta-catenin peptide was obtained using TRansfer Nuclear Overhauser Effect SpectroscopY (TRNOESY) experiments. Finally, we obtained the bound structure of the phosphorylated peptide showing the protons identified by STD NMR as exposed in close proximity to the molecule surface.

Ecdysteroid glycosides: identification, chromatographic properties, and biological significance.

Ecdysteroid glycosides are found in both animals and plants. The chromatographic behavior of these molecules is characteristic, as they appear much more polar than their corresponding free aglycones when analyzed by normal-phase high-performance liquid chromatography (HPLC), whereas the presence of glycosidic moieties has a very limited (if any) impact on polarity when using reversed-phase HPLC. Biological activity is greatly reduced because the presence of this bulky substituent probably impairs the interaction with ecdysteroid receptor(s). 2-Deoxy-20-hydroxyecdysone 22-O-beta-D-glucopyranoside, which has been isolated from the dried aerial parts of Silene nutans (Caryophyllaceae), is used as a model compound to describe the rationale of ecdysteroid glycoside purification and identification.