Ecdysteroid Conjugates Research Articles

Multicomponent Approach to Bioactive Peptide–Ecdysteroid Conjugates: Creating Diversity at C6 by Means of the Ugi Reaction

An efficient multicomponent protocol has been developed to access two different kinds of peptide–ecdysteroid conjugates. The approach exploits the reactivity of the 6-keto-7-ene group of 20-OH-ecdysone, allowing the unprecedented preparation of 6-amino and 6-isocyanide derivatives. The ecdysteroid derivatives were further employed in the Ugi reaction, together with formaldehyde and various structurally diverse carboxylic acids, isocyanides and amines, to afford a small family of potential multidrug-resistance modulators.

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.

Ecdysteroid levels in Daphnia magna during a molt cycle: Determination by radioimmunoassay (RIA) and liquid chromatography–mass spectrometry (LC–MS)

Despite their abundance and their enormous significance for various ecological processes, endocrine systems of microcrustaceans have been poorly investigated. Here, we used a highly sensitive radioimmunoassay (RIA) to determine free and conjugated ecdysteroid levels in whole-body extracts of adult Daphnia magna during a complete molt cycle. Ecdysteroid levels were predominated by free ecdysteroids. Starting from basal levels in the postmolt stage the concentration of free ecdysteroids increased sharply in the early premolt stage, followed by a sharp decline back to basal levels just prior to ecdysis. Polar and apolar ecdysteroid conjugates were found in rather low amounts with little variation during the molt cycle. Only small amounts of ecdysteroids were found in newly deposited eggs of D. magna, which suggest a sparing investment of maternal ecdysteroids into the eggs for early embryogenesis. As in whole-body extracts, free ecdysteroids were the predominant ecdysteroids found in eggs of D. magna, together with small amounts of polar and intermediary amounts of apolar conjugates. Hence, the pathways leading to polar and apolar ecdysteroid conjugates appear to be of minor importance in D. magna. Analyses of the immunodetectable peak in free ecdysteroids by liquid chromatography–mass spectrometry (LC–MS) revealed that molting is induced most probably by an increased level of 20-hydroxyecdysone. Microcrustaceans of the genus Daphnia are key components in freshwater food webs. Examination of the functional role of ecdysteroids in controlling developmental processes might help to understand the performance of the herbivorous grazer in its environment, in particular with regard to the adverse effects of environmental xenobiotics acting as endocrine disrupters.

On the distribution of phytoecdysteroids in plants.

The occurrence and levels of phytoecdysteroids in the seeds and other parts of plants grown from the seeds of 180 randomly selected plant species were assessed and compared. Ecdysteroids are frequently detectable in leaves and flowers, but less so in stems, roots and seeds. The seeds of 290 species were assessed for the presence of hydrolysable ecdysteroid conjugates. Low levels of conjugates were detected in a significant number of species, large amounts being present only when levels of free ecdysteroids were high. Individual plants of Arabidopsis thaliana were assessed for the presence of phytoecdysteroids. While plants of this species are generally ecdysteroid negative, individual plants in the population accumulate low levels of ecdysteroids. Extracts of seeds of 50 "ecdysteroid-negative" species were concentrated and partially purified to determine if they possess ecdysteroids at levels below the usual detection levels. Ecdysteroids were detectable by radioimmunoassay in almost all of these concentrated samples. Thus, all lines of evidence point to the conclusion that all species of plants have the capacity to produce at least low levels of phytoecdysteroids. This has important implications for the protection of crop species through enhancing ecdysteroid levels by breeding/genetic modification strategies.

Phytoecdysteroid profiles in seeds of Sida spp. (Malvaceae).

Procedures are presented for the assessment of the phytoecdysteroid profiles in small plant samples (ca. 25 mg), using seeds of Sida spp. as an example. The procedures are suitable for the analysis of minute or valuable samples and provide copious information for chemotaxonomic purposes. Methanolic extracts of the plant material, after partitioning against hexane, were separated by reversed-phase gradient HPLC monitored by PAD, RIA and bioassay. Aliquots of the fractions were also treated with Helix pomatia hydrolases, followed by RIA and bioassay, in order to assess the presence of hydrolysable ecdysteroid conjugates. Further information could also be obtained by separation of samples using normal-phase gradient HPLC. Among 11 species of Sida examined, seed extracts of S. acuta (= S. carpinifolia) and S. rhombifolia were found to contain significant amounts of ecdysteroids, seed extracts of S. filicaulis contained only moderate levels, whilst the remaining species showed no detectable levels of ecdysteroids. The ecdysteroid profiles of the extracts of the three positive species were significantly different, demonstrating that phytoecdysteroids have chemotaxonomic value in this genus.

Endogenous ecdysteroid levels and rates of ecdysone acylation by intact ovaries in vitro in relation to ovarian development in adult female crickets, Acheta domesticus.

Ecdysteroid titres have been determined in adult female house crickets (Acheta domesticus) in relation to reproductive maturation. Ecdysteroid levels in newly emerged adult females are low except in the gut and carcass, which probably reflects the remnants of the preecdysial ecdysteroid peak. Ecdysteroid levels in all compartments increase markedly once ovarian weight surpasses 10 mg. Apolar ecdysteroid conjugates (ecdysone 22-fatty acyl esters) predominate in ovarian tissue throughout ovarian maturation, but low levels of free ecdysteroid and polar conjugated ecdysteroids are also present. During this period, two peaks of ecdysteroids (mainly free and apolar conjugated ecdysteroids) are observed in the haemolymph, gut, and carcass compartments. The peaks in the haemolymph occur when the ovarian mass reaches 30 and 100 mg. The gut and carcass may be acting as sinks or sites of metabolism for the hormone released from the ovaries. The rate of ecdysone acylation by ovaries was found to be developmentally regulated, increasing from low levels in the immature ovaries of newly emerged females as the ovaries increase in size. A semiquantitative assay has been developed to identify compounds which inhibit the conversion of [3H]ecdysone into 22-fatty acyl [3H]ecdysone by ovaries in vitro. A number of ecdysteroids possessing a free hydroxyl group as C-22 as well as the side-chain stereochemistry of ecdysone effectively inhibit this conversion, probably by acting as competitive substrates. In the cases of 20-hydroxyecdysone and ponasterone A, it was clearly demonstrated that these compounds are converted to a mixture of C-22 fatty acyl esters. Several other compounds which have been suggested to affect ecdysteroid metabolism/mode of action in other systems were also tested for their effects on the acyltransferase activity of ovaries in vitro.

20-Hydroxyecdysone 2-β- d-glucopyranoside from the seeds of Xerophyllum tenax

Bioassay/RIA-directed HPLC purification of a MeOH extract of the seeds of Xerophyllum tenax yielded 20-hydroxyecdysone and 20-hydroxyecdysone 2-β- d-glucopyranoside, the latter being a novel natural product. The structures were elucidated primarily on the basis of 1D and 2D NMR spectroscopic evidence. The ecdysteroid agonist activity of these isolates was determined on the B II tumorous blood cell line of Drosophila melanogaster. ED 50 values for 20-hydroxyecdysone and 20-hydroxyecdysone 2-β- d-glucopyra-noside were found to be 8.0 × 10 −9M and 1.3 × 10 −4M, respectively. The significance of the presence of ecdysteroid conjugates in plants is discussed.

Ecdysteroids in relation to adult development and reproduction in femaleRhipicephalus appendiculatus (Acari; Ixodidae)

In view of the paucity of information on ecdysteroids during tick development, the profiles of the free ecdysteroids, together with the polar and apolar conjugates have been established by radioimmunoassay during development of adult females of the hard tick, Rhipicephalus appendiculatus. The free ecdysteroid titre increased sharply to a peak approximately 3 days post-engorgement, a day preceding beginning of oviposition. This titre decreased to a low level, which was maintained throughout oviposition. Although the titre of polar ecdysteroid conjugates was appreciably less than that of the free ecdysteroids during the peak, the general profile of such conjugates was similar to that of the free ecdysteroids. In the case of the apolar ecdysteroid conjugates, the titre increased simultaneously with production of free ecdysteroids, but was maintained at a relatively high level until the end of oviposition, when it sharply declined. The apolar conjugates were the predominant form of ecdysteroids present during most of oviposition. The free ecdysteroids as well as the polar and apolar conjugates were shown to contain 20-hydroxyecdysone accompanied by smaller amounts of ecdysone by high-performance liquid chromatography-RIA (HPLC-RIA) and gas chromatography/mass spectrometry (selected ion monitoring; GC/MS [SIM]). © 1996 Wiley-Liss, Inc.

Ecdysteroid titers during postembryonic development of Dermanyssus gallinae (Acari: Dermanyssidae).

Ecdysteroid titers in methanolic extracts of protonymphal and deutonymphal stages of the chicken mite, Dermanyssus gallinae (De Geer), were measured using radioimmunoassay. Highest titers of ecdysteroids were present 24 h after feeding. Further studies using high-performance liquid chromatography radioimmunoassay showed that ecdysteroid immunopositive material had the same retention time as 20-hydroxyecdysone and ecdysone. Ecdysteroid conjugates appear to be present in polar and apolar fractions.

Biosynthesis of ecdysteroids in Zea mays

The incubation of several radiolabelled substrates with Zea mays plants demonstrated the incorporation of radiolabels into ecdysteroid conjugates. Radiolabelled [ 14C]ecdysone and [ 14C]20-hydroxyecdysone, were biosynthesized from [2- 14C]mevalonic acid (MVA) in Z. mays. These ecdysteroids can be released from their conjugates by treatment with wheat germ acid phosphatase or mild acid hydrolysis. The C-24 reduced side chain sterols, lathosterol and cholesterol were endogenously biosynthesized from [2- 14C]MVA and were identified by 1H NMR. Lathosterol accounts for ca 0.5% of the total sterol composition, whereas cholesterol is present at 2.5%. However, lathosterol was consistently found to have a specific activity three to six times that of cholesterol. Incubation of [4- 14C]cholesterol with Z. mays leaves also demonstrated incorporation into a 20-hydroxyecdysone conjugate. Incubation of [22,23- 3H]ecdysone with Z. mays demonstrated incorporation into radiolabelled ecdysone diphosphate and ecdysone polyphosphate, which were cleaved upon acid phosphatase treatment. Z. mays biosynthesizes primarily ecdysteroid conjugates and does not appear to produce detectable levels of non-conjugated ecdysteroids.

Age dependency and tissue distribution of ecdysteroids in adult male crickets, Gryllus bimaculatus de Geer (Ensifera, Gryllidae)

Ecdysteroid titers were determined in tissues (gut plus Malpighian tubules, carcass tissue, fat body, muscles, haemolymph, accessory reproductive glands, and testes) of male adult crickets, Gryllus bimaculatus, during the first 20 days of adult life as well as in spermatophores. In all tissues, except testes, total ecdysteroid titers are high on the day of imaginal moulting and then drop more or less continuously until day 8 after moulting. Distinctly higher levels are found on day 12 and 18 as well. Freshly moulted males contain high quantities of polar ecdysteroid conjugates in the digestive tract, testes, accessory reproductive glands, and haemolymph. Apolar ecdysteroid conjugates are mainly detectable in carcass tissue and fat body, but also in the haemolymph during entire adulthood. Free ecdysteroids represent the domineering class of moulting hormones in the gut during all stages of adult life. The significance of cycling ecdysteroid concentrations during adulthood is discussed in relation to spermatophore production and development of male accessory reproductive glands.

Biosynthesis of ecdysone and ecdysone phosphates in spinach

The polar ecdysteroid conjugate, ecdysone phosphate (2β,3β,14α,22 R,25-pentahydroxy-7-en-6-one-3- phosphate) was identified in excised first leaves of spinach, where it is endogenously produced during 20- hydroxyecdysone biosynthesis. Radiolabelled [ 14C]ecdysone phosphate was isolated from several excised leaf assays and was hydrolysed with wheat germ acid phosphatase to yield [ 14C]ecdysone. Incorporation of ecdysone into excised first leaves followed by 32P exposure produced a compound with 32P activity, with chromatographic properties identical to those of the isolated [ 14C]ecdysone phosphate and upon hydrolysis released ecdysone. In spinach first leaves with active ecdysteroid biosynthesis, ecdysone is present at 0.004% of the total free ecdysteroid and contained 6% of the total radioactivity from [2- 14C]mevalonic acid (MVA). These biosynthetically active tissues also produce radiolabelled lathosterol, ecdysone-3-phosphate and 20-hydroxyecdysone. In biosynthetically inactive tissue (immature apical organs) no radiolabelled lathosterol, ecdysone-3-phosphate, ecdysone or 20-hydroxyecdysone was produced from [2- 14C]MVA despite an active biosynthesis of C 29-sterols. Several intermediate and end product ecdysteroids, when incorporated into excised first leaves of spinach produced conjugates which were readily cleaved by wheat germ acid phosphatase. The ecdysteroid pathway appears to be regulated by the presence of ecdysteroid substrates.

Ecdysteroids during embryogenesis of the house cricket, Acheta domesticus: Occurrence of novel ecdysteroid conjugates in developing eggs

The identity and titres of ecdysteroids during embryogenesis of the house cricket, Acheta domesticus, have been investigated by radioimmunoassay coupled to high-performance liquid chromatography (HPLC/RIA). It has previously been shown [Whiting and Dinan, J. Insect Physiol. 34, 625–631 (1988); Insect Biochem. 19, 759–765 (1989)] that the ecdysteroids present in newly-laid eggs are ecdysone (80 ng/g fr. wt) and a mixture of ecdysone 22-long-chain fatty acyl esters (732 ng ecdysone/g fr. wt). During the first 7 days of embryogenesis at 26°C, there is little change in the level or identity of the apolar conjugated ecdysteroids, but these increase 3-fold by day 10 and remain at this high level until the end of embryogenesis (13.5 days). Free ecdysteroid levels fluctuate during embryogenesis and there is a transition from ecdysone at the beginning of the embryological period to 20-hydroxyecdysone as the major free ecdysteroid at the end of embryogenesis. Novel ecdysteroid conjugates are increasingly formed as the embryo develops. These conjugates have unusual chromatographic properties, appearing polar on silicic acid, but apolar on reversed-phase. Evidence is presented, based on sensitivity to porcine liver esterase and Helix enzyme hydrolysis coupled with HPLC/RIA, that these conjugates are double conjugates consisting of ecdysone or 20-hydroxyecdysone esterified to a fatty acyl group at C-22 and to a neutral polar or negatively-charged group at another of the hydroxyls (possibly C-25).

Hormonal activity of ecdysteroids from certain asiatic plants

Ecdysone activity of ecdysteroids and ecdysteroid conjugates from plants has been determined in three species of insects, Sarcophaga bullata (Diptera), Galleria mellonella (Lepidoptera) and Dermestes vulpinus (Coleoptera). Standardized injection assays were made on teh ligatured last instar larvae, the hormonal activity was expressed in the common ED-50 units. The activity of most ecdysteroids falls within a narrow range of dosages which is characterized by 2–10 μg limits in Sarcophaga, 7–12 μg in Galleria and 17–42 μg −1 of larval of body mass in Dermestes. The largest deviation from this rule has been found with turkesterone, which contains a “corticoid-like” hydroxylic function at C-11. Turkesterone (11,20-dihydroxyecdysone) is 10-fold more effective than ecdysterone (20-hydroxyecdysone) in Sarcophaga and Dermestes while it is almost inactive in Galleria. The biologically most active ecdysteroid conjugates are the esters of benzoic acid. Small but definite activity has been found in certain acetonides, while ecdysteroid glucosides and polyacetylated analogues were completely inactive in all injection assays.

Inactive prothoracic glands in larvae and pupae of Aedes aegypti: Ecdysteroid release by tissues in the thorax and abdomen

We report that the tissue identified as the prothoracic gland in fourth instar Aedes aegypti does not release ecdysteroids. The thorax, without the prothoracic gland, released the same amount of ecdysteroids in vitro as the intact thorax. Ecdysteroids were also released in vitro from the abdomen. Thoracic and abdominal ecdysteroid release was observed in the pupa and in all larval instars. During the fourth instar, a single increase and decrease in the rate of ecdysteroid release was observed from both the thorax and the abdomen. The thoracic and abdominal tissues of both sexes release ecdysteroids synchronously and in equal amounts. Hemolymph ecdysteroid titers are correlated with thoracic and abdominal release of ecdysteroids in vitro. Ecdysteroids are released from all abdominal segments, and the same amounts of ecdysteroids are released from the dorsal and ventral halves of the abdomen. More free ecdysteroids are released in vitro from thoracic and abdominal tissues than are present in the tissues themselves. Additionally, incubation of tissue homogenates with enzymes that hydrolyze ecdysteroid conjugates fails to increase ecdysteroid recovery, suggesting that as yet unidentified cells in both the thorax and abdomen synthesize ecdysteroids de novo.

Nemertean ecdysteroids: Relationship to reproduction

Summary Ecdysteroids in non-reproductive, reproductive, and post-reproductive specimens of two nemerteans, Paranemertes peregrina and Pantinonemertes californiensis, were studied using radioimmunoassay (RIA). Ecdysteroid concentrations increased during the reproductive season in females of both species. These data suggest the possibility that ecdysteroids are involved in reproduction. Study of ecdysteroid profiles using reverse phase high-performance liquid chromatography (RP-HPLC) followed by RIA showed that the types and relative amounts of different ecdysteroid metabolites were similar between mature male and female Pan. californiensis. In addition, [3H]-ecdysone was injected into mature male and female Pan. californiensis. The resulting RP-HPLC profiles indicated that both sexes metabolized [3H]-ecdysone to similar products. Both sexes produced polar and apolar ecdysteroid conjugates as the major metabolites of [3H]-ecdysone, with only traces of [3H]-20-hydroxyecdysone.

Profile of embryonic ecdysteroids in the decapod crustacean,Macrobrachium rosenbergii

Summary The ecdysteroid composition of embryos of M. rosenbergii at various stages of embryogenesis has been studied by means of radioimmunoassay (RIA), high-performance liquid chromatography—RIA (HPLC-RIA) and gas chromatography/mass spectrometry (selected ion monitoring; GC/MS(SIM). In newly-laid eggs, only free ecdysteroids (ecdysone and 20-hydroxyecdysone) were detected, whereas eggs at an early stage of embryogenesis contained ecdysone and also low levels of apolar ecdysteroid conjugates. Mid stage embryonating eggs contained comparatively high levels of both free ecdysteroid (ecdysone, 18.3 ng/g) and apolar conjugates (29.9ng/g). Late embryos contained by far the highest overall level of ecdysteroid, indicating the ability of the developing embryo to synthesize ecdysteroid. In contrast to the other stages, late embryos contained 26-hydroxyecdysone, ecdysonoic acid and 20-hydroxyecdysonoic acid at relatively low levels and 2, 3-diacetylecdysone 22-phosphate at a comparatively high level (324.0 ng/g)....

Epidermis as the source of ecdysone in an argasid tick.

Various tissues excised from nymphs of the tick Ornithodoros parkeri at the time of epicuticle deposition were incubated in vitro. The medium from the incubation of salivary glands, coxal glands, synganglion, testis, midgut, and fat body associated with tracheal trunk showed little or no ecdysteroid immunoreactivity. Only medium from incubated integument contained ecdysteroids. The following evidence indicated that epidermal cells are the source of ecdysone: (i) when dorsal and/or ventral integuments were incubated separately, both produced ecdysteroid immunoreactive material during the course of incubation. As compared with the ecdysteroid content in the integument before incubation, the amount of ecdysteroids produced after a 24-h incubation increased 4- to 7-fold; (ii) enzymatic hydrolysis showed that neither highly polar ecdysteroid conjugates nor apolar conjugates were stored in the integument; (iii) histological and scanning electron microscope observations demonstrated that these excised integuments consisted of newly deposited epicuticle and epidermis as well as some fat body cells; (iv) HPLC RIA showed that the integument with associated fat body produced ecdysone and 20-hydroxyecdysone, while the integument produced only ecdysone after removing fat body. Presumably, ecdysone secreted by epidermis was converted into 20-hydroxyecdysone by fat body.

Metastable ions arising from pseudomolecular [M  H]− ions produced by fast‐atom bombardment negative‐ion mass spectrometry of ecdysteroids

Metastable ions arising from pseudomolecular [M-H]- ions produced by fast-atom bombardment negative-ion mass spectrometry of a range of free ecdysteroids, ecdysteroid conjugates and polar metabolites were investigated by means of linked scanning at constant B/E. Free ecdysteroids displayed daughter-ion spectra which allow 20-hydroxyecdysteroids and ecdysteroids lacking C-20 hydroxylation to be readily distinguished. The ejection of acetic acid from acetylated ecdysteroids was also readily detectable. Characteristic metastable-ion decomposition of ecdysteroid acids was not observed, presumably as a result of charge localization. High-mass daughter ions were also lacking in the case of phosphate conjugates.

Apolar conjugates of ecdysteroids are not used as a storage form of molting hormone in the argasid tick Ornithodoros moubata

AbstractFifth (last) instar nymphs of th e tick Ornithodoros moubata convert ingested 20‐hydroxyecdysone (20E) to apolar conjugates AP2, which are then converted to th e more polar conjugates API. Only small quantities of free hormone were transferred to th e hemolymph and the carcass within t h e first 2 days after the blood meal. The proportion of radiolabel in these two compartments was highest at the time of the endogenous ecdysteroid peak; however, no traces of free [3H]20E were detected. The conversion probably occurs principally in the intestinal cells. Eleven days after ingestion, 84% of the radiolabel is located in the digestive tract, mainly in the form of API conjugates.API obtained in second instar nymphs fed with [3H]ecdysone ([3H]E) remain stable throughout the following nymphal instars. The ecdysteroid moiety of APT remained unchanged. The hydrolysis, although not complete, always yielded a peak comigrating with the reference E but never 20E or any other clearly distinct peaks that may have corresponded to metabolites of 20E. Less label per individual was present in adults, but its nature remained the same, viz., API mainly located in the digestive tract. In females, 2.5% of the label was transferred to the progeny during the first ovipositional cycle.Apolar products (mainly AP2) that accumulated in eggs of females injected with [3H]E or [3H]20E during vitellogenesis remained unchanged during the whole embryonic development. During the molting cycle of larvae, there was only a slight conversion of AP2 to API, but esterase hydrolysis of these products released the same percentages of E and 20E as in the freshly laid eggs.We conclude that in this tick species apolar conjugates of ecdysteroids are inactivation metabolites that are not reutilized during the development of the animal. These metabolites are mainly retained in the tick, probably because of its peculiar blocked midgut. Several studies have shown that in other arthropod species (ticks, spiders, and insects), these apolar metabolites are excreted in the feces.