Research Update:Ecdysterone:20-hydroxyecdysone.:Introduction and Its Benefit Applications,ecdysteroid hormone,body building.Serie No.:Pure09.Pure Phytochemical Series.MDidea Extracts Professional.

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Research Update:Ecdysterone:20-hydroxyecdysone.

Phytoecdysteroid constituents from Cyanotis arachnoidea.:

Four phytoecdysteroids that have only 19 or 21 carbons, named 11alpha-hydroxyrubrosterone (1), dihydroxyrubrosterone (2), rubrosterone (3) and poststerone (4), were isolated from the whole plant of Cyanotis arachnoidea C.B. Clarke. Among them, 1 was a new compound. Their structures were elucidated by spectroscopic methods.

Chemical constituents of Cyanotis arachnoidea.:

AIM: To investigate the chemical constituents of Cyanotis arachnoidea. METHODS: By using chromatographic methods for separation and combination with spectral analysis, their chemical structures were determined. RESULTS: Six compounds were identified as ajugasterone C-20, 22-acetonide (1), 20-hydroxyecdysone-20, 22-acetonide (2), 22-oxo-ajugasterone C (3), 22-oxo-20-hydroxyecdysone (4), beta-sitosterol (5), daucosterol (6). CONCLUSION: Compound 3 is a new compound, 4 was a new natural compound.

A new phytosterone from Cyanotis arachnoidea.:

From Cyanotis arachnoidea C.B. Clarke, a new phytosterone named cyanosterone A (1), along with three known compounds--beta-ecdysone (2), ajugasterone C (3) and beta-sitosterol (4) were isolated. The structure of the new compound was determined as 3beta,14alpha,20R,22R-tetrahydroxy-5alpha-cholest-7-en-6-one on the basis of physicochemical properties and spectral analysis.

Regulation of Schistocerca gregaria neuroparsin transcript levels by juvenile hormone and 20-hydroxyecdysone.:

Neuroparsins (NPs) are small proteins that were originally discovered in the pars intercerebralis-corpus cardiacum neurosecretory complex of the migratory locust brain. From the desert locust, Schistocerca gregaria, we recently cloned four different transcripts, each coding for a distinct NP-related peptide. In addition to the brain, some NP-like precursor (Scg-NPP) transcripts also occur in a number of peripheral tissues, and their expression levels are controlled in a gender- and stage-dependent manner. Previous studies revealed a close correlation between Scg-NPP transcript levels and the gonotrophic cycle. In the present report, we demonstrate that certain Scg-NPP transcript levels are significantly altered upon injection of juvenile hormone (JH) or 20-hydroxyecdysone (20E) in adult gregarious desert locusts (five days after final ecdysis). While Scg-NPP1 transcript levels did not significantly change as a result of hormone treatment (animals were analyzed 24 h after injection), Scg-NPP2, Scg-NPP3, and Scg-NPP4 displayed hormone-dependent regulation in various tissues. Scg-NPP2 and Scg-NPP3 transcript levels significantly increased in the brain of JH-treated locusts. In addition, JH induction of Scg-NPP3 and Scg-NPP4 transcripts was observed in male fat body and in male and female gonads. Furthermore, 20E injection also induced Scg-NPP2, Scg-NPP3, and Scg-NPP4 transcripts in desert locust gonads. This is the first report showing NP-like precursor gene expression in insect ovaries. Our study indicates that the expression levels of some Scg-NPP transcripts are regulated by developmental hormones, suggesting a close correlation between NP expression and the endocrine control of the reproductive cycle.

The analysis and crystallographic characterization of 20-hydroxyecdysone.:

20-Hydroxyecdysone (20E) is an insect molting hormone that is also widely spread in various plants. Many chromatographic methods can be used to identify and/or determine 20E content in samples of biological origin and various spectroscopic methods serve to identify its structural elements. We have utilized X-ray crystallography to reveal the stereostructures of 20E. Our data demonstrates that 20E exists in two different crystalline forms that are both orthorhombic modifications. One form is homo-molecular, with a limited freedom of internal rotation of the side chain around the C23-C24 bond and the other, which is a clathrate formed with methanol and water, which minimize the conformational freedom of the side chain.

Ecdysteroid coordinates optic lobe neurogenesis via a nitric oxide signaling pathway.:

Proliferation of neural precursors in the optic lobe of Manduca sexta is controlled by circulating steroids and by local production of nitric oxide (NO). Diaphorase staining, anti-NO synthase (NOS) immunocytochemistry and the NO-indicator, DAF-2, show that cells throughout the optic anlage contain NOS and produce NO. Signaling via NO inhibits proliferation in the anlage. When exposed to low levels of ecdysteroid, NO production is stimulated and proliferation ceases. When steroid levels are increased, NO production begins to decrease within 15 minutes independent of RNA or protein synthesis and cells rapidly resume proliferation. Resumption of proliferation is not due simply to the removal of NO repression though, but also requires an ecdysteroid stimulatory pathway. The consequence of these opposing pathways is a sharpening of the responsiveness to the steroid, thereby facilitating a tight coordination between development of the different elements of the adult visual system.

Antioxidant effect of 20-hydroxyecdysone in a model system.:

Changes in the level of lipid free-radical oxidation in mitochondrial fraction at the presence of 20-hydroxyecdysone in 1, 2, 4, 6, 8 microM concentration with the help of a chemiluminescence (ChL) method were investigated in vitro. Statistically authentic reduction of ChL kinetic parameters--I(s) and tg alpha, at 20-hydroxyecdysone presence in concentration of 2 microM was found. 20-hydroxyecdysone administration in concentration 4 microM and more leads to the statistically authentic change of all four ChL parameters. The antioxidizing effect of 20-hydroxyecdysone was compared with action of such antioxidant as a hydroquinone. The higher activity of 20-hydroxyecdysone as an antioxidant in comparison with the hydroquinone was shown. For changes of all four measured kinetic parameters of ChL, concentration of the hydroquinone as much 2-fold than for 20-hydroxyecdysone is necessary. On the basis of our researches in the model system a conclusion was made that 20-hydroxyecdysone has an antioxidizing action on lipid free radical oxidation in mitochondrial fraction in dependents on concentration. 20-Hydroxyecdysone has antioxidizing properties directly, in these conditions in vitro, when its metabolites formation does not occur yet.

Induction of Sarcophaga central nervous system remodeling by 20-hydroxyecdysone in vitro.:

Proliferation and apoptosis of neural cells were found to be induced simultaneously when larval brains of Sarcophaga peregrina were cultured in the presence of 20-hydroxyecdysone (20-HE) for 24 h. The locations of proliferating cells and apoptotic cells in the brain hemispheres were different. The morphology of brains exposed to 20-HE for a short period proceeded to change sequentially when culture was continued for 2 days even in the absence of 20-HE. These changes mainly consisted of enlargement of the brain hemispheres and extension of the interval between two hemispheres, which closely paralleled the morphological changes of brains that occur in the early pupal stage, suggesting that ecdysteroid alone is sufficient to induce the remodeling of the central nervous system of holometabolous insects. Synthesis of a protein with a molecular mass of 66 kDa was shown to be selectively repressed when brains were cultured in the presence of 20-HE.

Effect of ecdysterone on glucose metabolism in vitro.:

The aims of this study was to investigate whether ecdysterone is able to exert glucose-lowering effect on hepatocytes or stimulate the secretion of insulin. HepG2 cell line was used for glucose consumption (GC) studies. At moderate high glucose concentration (11.1 mmol/L), GC of HepG2 cells was increased by 44% to 77% with ecdysterone 1 x 10(-6) to 1 x 10(-4) mol/L, which was comparable to that with 1 x 10(-3) mol/L metformin. The glucose-lowering effect of ecdysterone decreased as the glucose concentration of medium increased. The maximal potency was reached in the presence of 5.5 mmol/L glucose, and the effect was disappeared as the glucose consumption was increased to 22.2 mmol/L. This effect was independent on insulin concentration, which was similar to that of metformin and was different from that of troglitazone, whose glucose-lowering effect was insulin-dependent. Troglitazone had a better antihyperglycemic potency than metformin when insulin was added. Simultaneously, a significant toxicity of troglitazone to HepG2 cells was observed. betaTC3 cells were not stimulated by ecdysterone, that is, no secretogogue effect of ecdysterone was observed. The results indicate that ecdysterone is able to exert the glucose-lowering effect in hepatocytes which is insulin-independent, but has no effect on insulin release.

Neuroendocrine roles of the brain in the regulation of 20-hydroxyecdysone responsiveness in two types of diapause pupae of the cabbage armyworm, Mamestra brassicae.:

The cabbage armyworm, Mamestra brassicae, has winter-and aestival- diapause pupae (WD- and AD-pupae) showing differences in the strength of diapause. We tried to quantify diapause-strength by measuring the doses of 20-hydroxyecdysone (20-E) required to induce adult development in WD-, AD- and decerebrated non-diapause pupae (ND-pupae). The role of the brain in the regulation of diapause-strength was studied through the decerebration and brain-reimplantation of WD-and AD-pupae. The 20-E doses required for adult development were small within the first 2 days of pupation, and increased thereafter to reach a constant level about 10 days after pupation in AD- and decerebrated ND-pupae. The required 20-E doses in WD-pupae increased for more than 40 days after pupation. When 0-day-old WD- and 0-day-old AD-pupae were decerebrated, required 20-E doses increased after pupation and reached a constant about 10 days later. The required 20-E dose reached a constant level in decerebrated WD-pupae that was smaller than that observed for decerebrated ND- and WD-pupae. Furthermore, the required doses increased when 0-day-old WD-pupal brains were reimplanted into decerebrated WD- and decerebrated ND-pupae. In WD-, AD- and decerebrated ND-pupae, diapause-strength can be represented as the 20-E dose required for adult development. Diapause-strength is weak after pupation, increases thereafter, and reaches a constant about 10 days later in AD- and decerebrated ND-pupae. In WD-pupae, diapause-strength increases for more than 40 days after pupation and reaches a level that is twice that estimated for AD-pupae. Brains of diapausing WD-pupae may secrete a factor that suppresses the 20-E responsiveness of pupal organs, for the purpose of maintaining winter-diapause.

Effect of 20-hydroxyecdysone on cannibalism, sexual behavior, and contact sex pheromone in the solitary female spider.:

We investigated the effect of exogenously administered 20-hydroxyecdysone (20E) on sexual behavior and on contact sex signals of adult females of the spider Tegenaria atrica. In untreated or control females cannibalism occurred in 36-47% of encounters, whereas no cannibalism at all was observed with 20E-treated females. The frequency of sexual receptivity was low (42%) in untreated or control females and high (100%) in 20E-treated females. Variability of female sexual behavior could be related to quantitative changes in web and cuticular compounds. We showed that 20E altered the production of polar contact sex pheromones on web and cuticle: methylesters and fatty acids are produced in higher amounts in 20E-treated females. Bioassays demonstrated clearly that methylesters and fatty acids of webs and cuticles were involved in attraction of males. Moreover, bioassays with synthetic substances showed that four fatty acids (myristic, pentadecanoic, hexadecanoic, and oleic acids) and one methylester (methyl stearate) are particularly effective in eliciting sexual behavior in males. In T. atrica, 20E inhibits cannibalism during sexual activity and changes sex pheromone production.

Nerve growth factor expression response to induction agent booster dosing in transfected human embryonic kidney cells.:

To assess whether nerve growth factor (NGF) expression would respond to booster dosing with the inducing agent ponasterone A, human embryonic kidney cells (HEK-293) were transfected with human NGF cDNA. Cells were cultured for 5 days in media with or without ponasterone A. On day 5, controls received a ponasterone A media replacement, whereas experimental groups received ponasterone A booster media replacement. NGF protein expression bioactivity was assessed using a PC-12 cell bioassay and the concentration of secreted NGF was quantified using NGF enzyme-linked immunosorbent assay. Cells with and without ponasterone A were left for 5 days without changing the medium. On day 5, the supernatants were collected and flash-frozen for enzyme-linked immunosorbent assay. The ponasterone A-positive and -negative booster medium was replaced in the appropriate wells. Supernatants were collected from the wells at 2, 4, and 6 days after the booster dose and removal of original supernatant. The medium was flash-frozen for enzyme-linked immunosorbent assay (1.5 ml), and the remaining 500 mul was transferred to PC-12 cells seeded onto 12-well plates to determine NGF bioactivity. All experiments were performed in quadruplicate. NGF production was measured daily by enzyme-linked immunosorbent assay over a 6-day period after the ponasterone A booster to a maximal release of 1233 +/- 130 pg/ml at day 6 (11 days after original induction). Maximal NGF production per 10(3) cells was 2.5 +/- 0.61 pg at day 6. Bioactivity was determined by percentage differentiation (per 100 cells counted) at 26, 52, and 98 percent for ponasterone A-treated wells on 2, 4, and 6 days after booster dosing (7, 9, and 11 days after induction), respectively. PC-12 cell differentiation was not visualized in the ponasterone A-negative control wells. Human NGF-EcR-293 cells can inducibly secrete bioactive NGF when exposed to the induction agent ponasterone A. Furthermore, repeated bioactive NGF expression peaks beyond that previously demonstrated can be achieved using induction agent booster dosing, indicating the ability to regulate the system over an extended period.

The ecdysone regulatory cascade and ovarian development in lepidopteran insects: insights from the silkmoth paradigm.:

The developing ovariole of the silkmoth Bombyx mori represents an excellent model for studies on the changes that occur in gene expression during the execution of long-term developmental programs. All stages of follicle development, which differ from each other by 2-2.5 h of developmental time, are organized in a single array and can be isolated simultaneously for physiological, biochemical and gene expression studies from a single animal. Recently, significant progress has been made toward the understanding of the hormonal regulation and autonomous implementation of the developmental program that governs follicular cell differentiation during oogenesis in Bombyx. In this review, the developmental career of the ovarian follicle during pharate adult development is discussed in view of the new physiological, biochemical and gene expression data that have recently accumulated. An overview is presented of the changes in expression of structural and regulatory genes and their hormonal regulation in the developing follicle during the transitions among the broad developmental periods of previtellogenesis, vitellogenesis and choriogenesis.Ovarian development in silkmoth pharate adults is induced by 20-hydroxy-ecdysone (20E) through the activation of a regulatory cascade similar to the one observed during Drosophila development. The transition from previtellogenesis to vitellogenesis corresponds to a late response to the hormone and is characterized by the induction of the expression of a unique isoform of the nuclear receptor BmHR3 and the follicular cell-specific yolk protein ESP. The transition from early to middle and late vitellogenesis and choriogenesis, on the other hand, is regulated by positively and negatively acting intra- and extra-ovarian factors. In vitro cultures of developing ovarioles reveal the requirement for the presence of an as yet unidentified growth factor(s) in the hemolymph, while the follicle developmental arrest that is observed after treatment with the ecdysone agonist tebufenozide indicates the requirement for a decline in the level of 20E. The initiation of choriogenesis is characterized by the transcriptional activation of the gene BmGATAbeta that encodes GATA transcription factors, and the chorion genes in the follicular cells. Furthermore, modulation of the activity of BmGATAbeta at the posttranscriptional level is crucial for the stage-specific activation of chorion genes during late choriogenesis.The developing ovariole of the silkmoth is, therefore, emerging as an important model for the study of insect oogenesis and the action of the steroid hormone 20E at the molecular level.

Differential expression and regulation by 20-hydroxyecdysone of mosquito ecdysteroid receptor isoforms A and B.:

Cloning of the AaEcR-A isoform, along with the previously cloned AaEcR-B isoform, has permitted us to evaluate the expression of AaEcR isoforms during mosquito vitellogenesis. Mosquito EcR isoform transcripts exhibited dramatically different patterns of expression after a blood meal-triggered activation of vitellogenesis in the fat body. The AaEcR-B transcript level rose sharply by 4-h post blood meal (PBM), coinciding with the small ecdysteroid peak, and then declined reaching its lowest level at 16-24-h PBM. In contrast, the AaEcR-A transcript peaked at 16-20-h PBM, coinciding with the large ecdysteroid peak. AaEcR-B and AaEcR-A transcripts exhibited a striking difference in sensitivity to 20-hydroxyecdysone (20E), being maximally activated at 10(-8) and 10(-6) M, respectively. Both ecdysteroid receptor (EcR) isoform mRNAs were transcribed in a cycloheximide-independent manner, suggesting that they are direct targets of 20E. However, AaEcR-A transcription requires continuous presence of 20E, while AaEcR-B mRNA level rose for 4 h and then declined under the same conditions. These results indicate the mosquito EcR isoforms play distinct physiological functions during vitellogenesis in the mosquito fat body.

Vitamin D3 and 20-hydroxyecdysone -- inhibitors of free radical lipid oxidation during D-hypervitaminosis.:

Under D-hypovitaminosis (control) conditions the statistically reliable increase of blood serum lipids free radical oxidation was revealed in comparison with the intact animals. Administration of vitamin D3 to the animals suffering from D-hypovitaminosis leads for statistically reliable decrease of blood serum lipids free radical oxidation, while 20-hydroxyecdysone in quantity of 0.02 mg per 1 kg of the animal body weight displays the antioxidative properties. Its antioxidative effect is characterized by a statistically reliable increase of Tind chemiluminescence kinetical parameter as compared with the control. Under D-hypovitaminosis conditions in the mitochondrial membranes the products of lipids free radical oxidation--dien conjugates are accumulated. In the case of administrating to the animals suffering from D-hypovitaminosis D3 or 20-hydroxyecdysone these oxidation products are absent. 20-Hydroxyecdysone under these conditions have been revealed as inducing accumulation in the mitochondrial and microsomal membranes of the substances with lambda 225 nm.

Photochemical transformation of 20-hydroxyecdysone: production of monomeric and dimeric ecdysteroid analogues.:

Structural modification of 20-hydroxyecdysone (20E) based on photochemical transformation yielded dimeric ecdysteroid 7alphaH,7'alphaH-bis-[(20R,22R)-2beta,3beta,20,22,25-pentahydroxy-5beta-cholest-8(14)-en-6-one-7-yl] as a main product. Its structure was determined by detailed NMR analysis. Furthermore, two new monomeric analogues: 14-epi-20-hydroxyecdysone and 14-deoxy-14,18-cyclo-20-hydroxyecdysone were identified in addition to the earlier described 14-deoxy and 14-hydroperoxy derivatives of 20E. Formation of the specific and so far unique ecdysteroid dimer has not been observed in earlier photo-transformation studies. The transformed dimeric analogue of 20-hydroxyecdysone retained the high agonistic activity on the ecdysone receptor in the B(II)-bioassay compared with the original 20E.

Study of excretion of ecdysterone in human urine.:

A study of excretion in human urine of ecdysterone, which is the active component of several over-the-counter supplements such as "Ecdysten", reportedly used by athletes, is presented. The study was performed after oral administration of 20 mg of ecdysterone. The collected urine samples were prepared using the standard screening extraction procedure for the free and conjugated fraction of anabolic steroids, and analyzed by gas chromatography (GC) coupled with quadrupole mass spectrometry (MS) and also with high-resolution mass spectrometry (HRMS). Two ecdysterone metabolites were identified and detected along with unchanged ecdysterone. Accurate mass measurements were made for diagnostic ions, including the molecular ion of the main metabolite of ecdysterone, deoxyecdysone, which, to our knowledge, has not previously been reported in the literature. These accurate mass measurements support the proposed fragmentation scheme.

20-hydroxyecdysone causes increased aggressiveness in female American lobsters, Homarus americanus.:

Lobsters become transiently more aggressive before ecdysis. This aggressiveness accompanies an increase in hemolymph titers of 20-hydroxyecdysone (20-HE). Combats between intermolt female lobsters, injected with premolt levels of 20-HE, and larger, saline-injected opponents were videotaped. Aggressive, defensive, and avoidance behaviors were ranked according to aggressiveness in a Rank of Aggression hierarchy, which included opponent-directed and (nonopponent) redirected behaviors. Treated animals performed more and more highly aggressive behaviors than saline-injected controls. Opponents of treated animals performed fewer aggressive behaviors than saline-injected control opponents. Controls performed more defensive behaviors than treated animals, when redirected behaviors were considered. Differences in avoidance behaviors among the four types of combatants were not significant. The total aggressive content was the same in treated and control fights, but the interactions between combatants in the two fights were significantly different. Treated animals were equally as aggressive and defensive as their opponents; controls were relatively less aggressive and more defensive than their opponents. These results correlate with molt-cycle variations in behavior, 20-HE titers, and the effects of 20-HE and molt-differentiated hemolymph on the claw opener muscle. They suggest that 20-HE orchestrates intrinsic, cellular, and nuclear events that produce the molt-cycle transformations in agonistic behavior and aggressive state of lobsters

Determination of ecdysterone in Achyranthes bidentata Bl. and its activity promoting proliferation of osteoblast-like cells.:

AIM: To study the activity of ecdysterone from Achyranthes bidentata Bl. (AB) promoting proliferation of osteoblast-like (OB-like) UMR106 cells and to determine its content in AB by HPLC method. METHODS: Ecdysterone isolated from AB was cultured with OB-like cells UMR106 together in vitro and the proliferation of OB-like cells was determined by MTT assay. The chromatographic conditions for determining ecdysterone included an ODS column (250 mm x 4.6 mm, 5 microns), a mobile phase consisting of a mixture of water-acetontrile-tetrahydrofuran (86:11:3), detection wavelength of 243 nm, and column temperature of 27 degrees C. Phenacetin was used as the internal standard. RESULTS: The ecdysterone from AB had significant activity promoting proliferation of OB-like cells, the proliferation was promoted by 41% (n = 3). The average recovery of ecdysterone was 96.2% (RSD = 2.1%), the calibration was linear in the range of 30-300 micrograms.mL-1 (gamma = 0.9998). CONCLUSION: Ecdysterone was screened quickly by cultivating with OB-like cells together in vitro. The HPLC method is accurate, fast and reproducible for the determination of ecdysterone in AB.

20-Hydroxyecdysone release from biodegradable devices: the effect of size and shape.:

20-Hydroxyecdysone (20-OH) is a natural compound with many demonstrated effects on the physiological functions of vertebrates, particularly increased protein synthesis. Our study sought a suitable dosage form with continuous release of the drug lasting several weeks for implantation into agricultural animals. Biodegradable microparticles and implants of poly(L-lactic) and poly(DL-lactic) acids were prepared. Oligomers of these materials were synthesized, and a method of melting the binary mixture of the oligomer and 20-OH was employed. The particles were prepared simply by grinding the solidified block of the melt and sieving. Implants were prepared by extruding the melt into silicone tubes, removing the solidified content, and cutting into cylinders of 2 mm diameter and various lengths. A new method of preparation of hollow cylinders by aspirating air into silicone tubes filled with the melt was developed. The experiments demonstrated stability of 20-OH during heat treatment. Release of the active ingredient was tested in static in vitro conditions, analogous to those at the site of implantation, and prolonged drug release was obtained with both types of implant. The hollow implants gave release rates nearest to ideal zero-order kinetics and would appear most appropriate for testing in vivo.

Enhancement of GABA-mediated inhibition of rat medial vestibular nucleus neurons by the neurosteroid 20-hydroxyecdysone.:

In vivo electrophysiological and patch-clamp studies were performed to determine whether 20-hydroxyecdysone (20-HE), a neurosteroid, influenced neuronal activities of the medial vestibular nucleus (MVN) using chloral hydrate-anesthetized rats and dissociated MVN neurons, respectively. Single neuronal activities of MVN were extracellularly recorded with a glass-insulated silver wire microelectrode attached along a seven-barreled micropipette. Each micropipette was filled with 20-HE, glutamate, bicuculline or 2 M NaCl. These chemicals were applied microiontophoretically to the immediate vicinity of the target neurons. Microiontophoretically applied 20-HE (20-80 nA) dose-dependently decreased rotation-induced firings of both type I and II neurons, which were identified according to their responses to horizontal sinusoidal rotations. Microiontophoretically applied bicuculline, a GABAA receptor antagonist, inhibited 20-HE-induced decreases in neuronal firing of MVN. These findings suggest that 20-HE potentiates the action of GABA, probably by acting directly on the GABAA receptor of MVN neurons. In addition, microiontophoretically applied 20-HE decreased firings induced by glutamate in both type I and II neurons. This decrease by 20-HE was also antagonized with bicuculline. Furthermore, the effects of 20-HE on GABA-induced currents in acutely dissociated MVN neurons were investigated using the whole-cell patch-clamp technique. Under voltage-clamp conditions, GABA (10 microM)-induced currents were potentiated in the presence of 20-HE (100 microM). These findings suggest that 20-HE inhibits MVN neurons by acting on the modulatory site on GABA receptor-ion channel complexes to potentiate GABA inhibition.

Effects of vitamin D3 and ecdysterone on free-radical lipid peroxidation.:

Free-radical-induced lipid peroxidation was studied in vivo by measuring chemiluminescence of tissues from vitamin D-deficit animals before and after peroral administration of low-molecular-weight biological steroids vitamin D3 (cholecalciferol) and ecdysterone. The kinetics of lipid chemiluminescence in model systems in vitro were determined in blood serum and microsomal and mitochondrial fractions of the liver. Vitamin D3 (cholecalciferol) and ecdysterone displayed antiradical properties; the latter was more potent in this respect. Oxidation of lipids by hydroxyl groups contained in ecdysterone can account for its antiradical effect. Ecdysterone and D3 may cause antiradical effects through the same mechanisms.

Effect of vitamin D3 and 20-hydroxyecdysone on the content of ATP, creatine phosphate, carnosine and Ca2+ in skeletal muscles.:

The effect of vitamin D3, 20-hydroxyecdysone and extract from Serratula coronata containing 20-hydroxyecdysone on the level of basic metabolites in the skeletal muscles of rats has been studied. It was shown that development of D-hypovitaminosis is accompanied by the decrease in content of ATP, creatine phosphate, carnosine, and by the increase of Ca2+ content. Against the background of D-hypovitaminosis the 20-hydroxyecdysone and the extract from Serratula coronata which contains 20-hydroxyecdysone promote the increase of the amount of these metabolites up to the control of one and normalize Ca2+ content in them.

8-O-acetylharpagide is a nonsteroidal ecdysteroid agonist.:

We have identified a novel nonsteroidal ecdysteroid agonist. This compound was isolated from a methanol extract of Ajuga reptans L. (Lamiaceae) and the structure was identified by spectroscopic methods as 8-O-acetylharpagide. We have characterised this compound as an ecdysteroid agonist in a transactivation assay using beta-galactosidase as the reporter gene regulated by ecdysteroid response elements. In this assay, 8-O-acetylharpagide has an EC50 of 22 microM. The compound also competes with tritiated-ponasterone A for binding to the Drosophila ecdysteroid receptor. Finally, it induces differentiation of Drosophila Kc cells as would be expected of an ecdysteroid agonist. This iridoid glycoside is common to several plant species and may play a role in the natural defense mechanisms of plants.

Characterization of the dopa decarboxylase gene of Manduca sexta and its suppression by 20-hydroxyecdysone.:

Manduca dopa decarboxylase (DDC) cDNA was isolated, sequenced, and found to be most closely related to Drosophila DDC (72% amino acid identity). Culture of Day 2 fourth instar larval epidermis with 20-hydroxyecdysone (20E) showed that 20E was necessary to determine the later expression of the gene, but its removal was required for this expression to occur. Experiments with the protein synthesis inhibitors, cycloheximide and anisomycin, and the mRNA synthesis inhibitor, alpha-amanitin, showed that 20E induced a protein(s) which suppressed transcription of the DDC gene. Gel mobility shift assays using epidermal extracts and various fragments of the first 1.1 kb of the 5' flanking region of the DDC gene showed only one DNA fragment 87 to 167 bp upstream of the 5' initiation site that bound a nuclear protein(s) with the expected developmental specificity. The protein was abundant at the time of high ecdysteroid titer when no DDC mRNA was present, but low both before the rise (no DDC mRNA) and after the decline of ecdysteroid titer (maximal DDC mRNA). Thus, this protein(s) is a candidate for an ecdysteroid-induced transcription factor which acts to suppress DDC transcription. DNase I footprinting assays confirmed by use of a specific oligonucleotide showed that this protein(s) bound to the sequence 5'-GGCTTATGCGCTGCA-3'.

Potentiation of GABA-induced inhibition by 20-hydroxyecdysone, a neurosteroid, in cultured rat cortical neurons.:

Effects of 20-hydroxyecdysone (20-HE), a neurosteroid, on cultured rat cortical neurons were examined using the whole cell recording technique. Under the voltage and current clamp conditions, brief application (5 sec) of 20-HE alone did not produce current changes nor any changes in the membrane potential. However, the chemical dose-dependently potentiated the GABA-induced current and hyperpolarization, which were blocked by bicuculline. These results suggest that 20-HE acts on the modulatory site of the GABAA receptor and potentiates GABAergic inhibition in rat cortical neurons.

Mechanism of action of ecdysterone in chickens with D-hypovitaminosis. Early phase of effect.:

Early action of ecdysterone in enterocytes of thin intestine of chickens with experimental D-hypovitaminosis has been studied with the aim to find out molecular mechanisms of its action. Changes in biosynthesis of nucleic acids accompanied with normalization of Ca translocation, biosynthesis of proteins, lipids and nucleic acid precursors (purine and pyrimidine nucleotides) as well as oxypurines were observed over the time interval (0-2 h) after ecdysterone administration. Multifunction character of the early action of ecdysterone indicates to the intermediary mechanism of gene activation by ecdysterone and provides for the availability of the secondary mediator for realization of its biological action in enterocytes.

Effects of the steroid hormone, 20-hydroxyecdysone, on the growth of neurites by identified insect motoneurons in vitro.:

During metamorphosis in the hawkmoth, Manduca sexta, identified larval leg motoneurons survive the degeneration of their larval targets to innervate new muscles of the adult legs. The dendrites and axon terminals of these motoneurons regress at the end of the larval stage and then regrow during adult development. Previous studies have implicated the insect steroid, 20-hydroxyecdysone (20-HE), in similar examples of dendritic reorganization during metamorphosis. The present studies were undertaken to test whether 20-HE acts directly on the leg motoneurons to regulate dendritic growth. Larval leg motoneurons were labeled with a fluorescent dye to permit their identification in culture following the dissociation of thoracic ganglia at later stages of development. Leg motoneurons isolated from early pupal stage animals (just before the normal onset of dendritic regrowth) survived in vitro and grew processes regardless of whether 20-HE was added to the culture medium. The extent of process outgrowth, however, as measured by the total length of all processes and the number of branches, was significantly greater for motoneurons maintained in the presence of 20-HE. The enhancement could be blocked by the addition of a juvenile hormone analog. By contrast, larval leg motoneurons that were isolated just before the normal period of dendritic regression did not show enhanced growth of neurites in the presence of 20-HE. The results suggest that 20-HE acts directly on the leg motoneurons to regulate the growth of processes during metamorphosis.

Immunomodulating effect of ecdysterones.:

Ecdysterone, its 20-desoxy-derivative alpha-ecdysone, their 2-desoxy-derivatives ecdysterone 2, 3, 22-triacetate and preparation BTI-4 have been studied for their effect on [3H]-thymidine incorporation in different populations of animal and human lymphocytes. It is shown the ecdysteron and its analogs in concentrations of 10(-12)-10(-5) M take considerable stimulating effect on DNA biosynthesis in animal lymphocytes activated by polyclonal mitogens. The concentration of ecdysterone being increased to 10(-4) m one can observe complete inhibition of activating effect of polyclonal mitogens. Effect of the studied ecdysteroids did not considerably depend on their structure. In case of splenocytes the stimulating effect of ecdysterone on DNA biosynthesis is less expressed than in the case of activated thymocytes. Ecdysterone was established to have considerable inhibiting effect on DNA biosynthesis in the culture of activated Con A cells of lymphocytes in the peripheral blood of healthy donors.

Ecdysterone regulatory elements function as both transcriptional activators and repressors.:

A synthetic, 23-bp ecdysterone regulatory element (EcRE), derived from the upstream region of the Drosophila melanogaster hsp27 gene, was inserted adjacent to the herpes simplex virus thymidine kinase promoter fused to a bacterial gene for chloramphenicol acetyltransferase (CAT). Hybrid constructs were transfected into Drosophila S3 cells and assayed for ecdysterone-inducible CAT expression. In the absence of ecdysterone a tandem pair of EcREs repressed the high constitutive level of CAT activity found after transfection with the parent reporter plasmid alone. After hormone addition very high levels of CAT activity were observed. Insertion of the EcRE pair 3' of the CAT gene also led to high levels of ecdysterone-induced CAT expression, but the repression of high constitutive levels of CAT activity failed to occur. The EcRE-CAT construct was cotransfected with plasmids containing tandem 10-mers or 40-mers of the EcRE but lacking a reporter gene. These additional EcREs led to a reduced level of ecdysterone-induced CAT activity and to an elevation of basal CAT activity in the absence of hormone. The data suggest that the receptor binds to the EcRE in the absence of hormone, blocking basal transcription from a constitutive promoter. In the presence of ecdysterone, receptor-hormone binding to the EcRE leads to greatly enhanced transcription.

Effect of ecdysterone on histamine release from rat peritoneal mast cells.:

Ecdysterone dose-dependently inhibited anti-IgE-induced histamine release from mast cells. Moreover, the rate and extent of histamine release from mast cells induced by Concanavalin A (Con A) are significantly diminished in samples incubated with ecdysterone. Ecdysterone inhibited both the initial and gradual rise in fluorescent response by anti-IgE and Con A. The effects of ecdysterone on the fluorescence response was correlated with the inhibition of histamine release. These results suggest the possibility that the inhibition of histamine release from rat mast cells by ecdysterone might be due to inhibition of Ca2+ mobilization from intracellular Ca2+ storage.

Effect of 20-hydroxyecdysone on the salivary glands of the male tick, Amblyomma hebraeum.:

Female ticks (Acari: Ixodidae) feed only once in the adult stage, dying after laying a large batch of eggs. During the early post-engorgement stage, haemolymph ecdysteroid titre rises, which is probably responsible for autolysis of the salivary glands that takes place at this time. Males, on the other hand, can re-attach and feed numerous times during the adult stage. Males were fed on rabbits for either 7 or 14 days. Haemolymph was collected either the day of removal from the host or 4 days later, and ecdysteroid titre was measured by radioimmunoassay. The approximate titre in all 4 groups was 20 ng of 20-hydroxyecdysone (20-OHE) equivalents/ml haemolymph. Fluid secretory competence in vitro can be used as an index of salivary-gland degeneration. The glands dissected from fed males which had been left off the host for 4 days lost 62% of their fluid secretory competence compared to glands dissected shortly after the males were removed. This loss in fluid secretory competence was reversed by allowing ticks left off the host of 4 days to resume feeding. Male salivary glands lost fluid secretory competence when exposed for 4 days in organ culture to 20-OHE; the effect was maximal at the lowest concentration tested (20 ng/ml). Thus, although male salivary glands were highly sensitive to 20-OHE, it is still not clear whether this hormone causes the tissue to degenerate.

New possibilities in searching for immunomodulators among compounds with a steroid structure.:

It was shown that a phytosteroid ecdisterone which is the principle of a new tonic drug ecdisten in doses of 5-20 mg/kg is able to stimulate the primary immune reaction slightly effecting the indices of T-cell immunity activity and phagocyte functions. On increasing ecdisterone dose to 50 mg/kg inhibition of the number of antibody cells in the mouse spleen was marked.

Ecdysteroids affect in vivo protein metabolism of the flight muscle of the tobacco hornworm (Manduca sexta).:

Ecdysteroid growth promotion of the dorsolongitudinal flight muscle of Manduca sexta was studied by measuring in vivo protein metabolism using both "flooding-dose" and "non-carrier" techniques. These procedures differ in that the former method includes injection of non-labelled phenylalanine (30 micromoles/insect) together with the [3H]amino acid. Injected radioactivity plateaued in the haemolymph within 7 min. With the flooding-dose method, haemolymph and intramuscular specific radioactivities were similar between 15 min and 2 h. Incorporation of [3H]phenylalanine into muscle protein was linear with either method between 30 and 120 min. Fractional rates (%/12 h) of synthesis with the flooding-dose technique were best measured after 1 h because of the initial delay in radioactivity equilibration. Estimation of body phenylalanine turnover with the non-carrier method showed 24-53%/h which was negligible with the flooding-dose method. Since the two methods yielded similar rates of protein synthesis, the large injection of non-labelled amino acid did not alter the rate of synthesis. Because the flooding-dose technique requires only a single time point measurement, it is the preferred method. The decline and eventual cessation of flight-muscle growth was mostly a consequence of declining protein synthesis though degradation increased between 76-86 h before eclosion and was relatively rapid. This decline in muscle growth could be prevented by treating pupae with 20-hydroxyecdysone (10 micrograms/insect). Protein accretion was promoted by a decline of up to 80% in protein breakdown, which was offset in part by a concurrent though much smaller decrease in protein synthesis. Therefore, ecdysteroids may increase flight-muscle growth by inhibiting proteolysis.

The effect of nerobol and ecdysterone on insulin-dependent processes linked normally and in insulin resistance.:

The effect of substances with anabolic activity (metandienone and ecdysterone phytoecdysteroid) on the manifestation of insulin effects was studied on a model of insulin resistance in rats induced by injections of hydrocortisone or by insulin insufficiency caused by alloxan. The sensitivity of the body to i. v. infusion of insulin and the reactivity of isolated fatty tissue to the hormone were increased after administration of these substances to test animals. The above effects of steroids were determined by nonspecific synthesis of total proteins in cells rather than by an increase in insulin secretion.

Ecdysteroids in vitro promote differentiation in the accessory glands of male mealworm beetles.:

Physiological peak doses of 20-hydroxyecdysone were added to organ cultures of young pupal accessory glands of male Tenebrio molitor. During subsequent culture in vitro or in vivo, the glands accumulated adult-specific antigens. Control organ cultures showed no such antigen accumulation. In this system, ecdysteroid controls not only cell cycles but also differentiation.

Ecdysteroid levels during embryogenesis in the shrimp, Palaemon serratus (Crustacea Decapoda): quantitative and qualitative changes.:

During the embryogenesis of Palaemon serratus profound changes in ecdysteroid concentrations were found. Ecdysteroid concentrations increase at the appearance of the Y-organ and a slight decrease is observed just before hatching. Two peaks of ecdysteroid concentration occur between these two events if Palaemon is reared at 19 degrees, but only one broad maximum is observed after rearing at 11-12 degrees. The pattern of ecdysteroids changes during embryogenesis; the ecdysone level decreases, while the 20-OH-ecdysone concentration and the amount of high polarity products increase. During the embryonic stage C the ratio of 20-OH-ecdysone to ecdysone was shown by HPLC analysis to steadily increase. After enzymatic hydrolysis of high-polarity products the main RIA-positive material elutes from reversed-phase HPLC columns as 20-OH-ecdysone.

Differential responses of the dopa decarboxylase gene to 20-OH-ecdysone in Drosophila melanogaster.:

The dopa decarboxylase gene (Ddc) of Drosophila melanogaster responds to 20-OH-ecdysone in the mature larval epidermis and in imaginal discs (presumptive adult epidermis) in a tissue-specific manner. Exposure of the mature larval epidermis to 20-OH-ecdysone caused a rapid accumulation of DDC transcripts. In the absence of protein synthesis, transcript accumulation was substantially reduced suggesting an indirect hormonal effect on DDC transcription (and/or RNA turnover). By contrast, neither DDC activity induction nor transcript accumulation was detected in imaginal discs cultured in the continuous presence of the hormone. However, when discs were exposed to 20-OH-ecdysone and then cultured in its absence, DDC activity and DDC transcript levels started to increase 6 hr after hormone withdrawal. A Northern analysis failed to reveal any novel transcripts in discs making the utilization of an alternative promotor an unlikely explanation for the very different responses of the Ddc gene in the two epidermal tissues. The results demonstrate that the Ddc gene in the larval epidermis responds rapidly to an increase in hormone titer. In imaginal discs a fall in hormone titer is required before DDC transcripts accumulate.

Histochemical analysis of the ecdysterone-regulated expression of the Drosophila genes P1 and LSP-2.:

3H-labeled DNA probes for the ecdysterone-inducible Drosophila genes P1 and LSP-2 were hybridized in situ to RNA in sections of embryos and larvae. Intense hybridization was detected specifically in fat body cells of third-instar larvae and not in other cells of third-instar larvae nor in any cells at earlier stages. These results confirm the stringent tissue- and stage-specificity of P1 and LSP-2 expression. Hybridization of both probes occurred to virtually all the cells in the fat bodies, indicating that both genes are expressed in the same cells. Since P1 expression begins several hours later than LSP-2 expression, and appears to be induced directly by ecdysterone, this finding implies that one or more of the fat body components mediating the response to ecdysterone is gene-specific.

Alterations in rat lipid metabolism following ecdysterone treatment.:

The influence of ecdysterone on the lipid metabolism in liver and adipose tissue from rat was investigated using 14C-acetate and 32P-orthophosphate as precursors. Ecdysterone produced an increase in 14C-acetate incorporation into triglycerides. A concomitant decrease in free fatty acids and diglycerides was observed. The effect of ecdysterone on triglyceride lipase activity was investigated and a significant decrease was found. Ecdysterone produced a significant increase in the specific activity of phosphatidylethanolamine and phosphatidylserine in liver. On the contrary, the specific activity of phosphatidylcholine was reduced. In adipose tissue, the most evident effect observed was the increase of specific activity of phosphatidylcholine. These results contribute to knowledge of the heterophylic action of ecdysterone.

A novel form of DOPA decarboxylase produced in drosophila cells in response to 20-hydroxyecdysone.:

Two cloned derivatives of the Kc cell line of Drosophila were shown to produce DOPA decarboxylase following administration of the steroid moulting hormone 20-hydroxyecdysone. In the continuous presence of the hormone at a concentration of 2 X 10(-7) M, DOPA decarboxylase activity first appeared between 48 and 72 h. Because of this lag, the tissue culture system promises to serve as a useful model for those in vivo situations where increases in the hormone titre precede increases in DOPA decarboxylase activity. In clone 7C4, after maximal enzyme activity was achieved at 144 h, the enzyme activity per cell decreased as the cells resumed division following the hormone-induced division arrest. In clone 7E10, cell division never resumed in the presence of 20-hydroxyecdysone and DOPA decarboxylase activity per cell increased continuously from the time it first appeared. When line 7E10 was exposed to a 6-h pulse of the steroid, enzyme activity appeared about 18 h earlier than in the presence of continuous hormone and, further, the cells were released from division arrest. Enzyme activity per cell then declined from an early 96-h maximum. The enzyme produced by the cell lines was immunologically distinct from the enzyme produced in vivo and ion-exchange column chromatography resolved the enzyme from cells and intact organisms into two species.

  Scientific References:

  1.Research Update:Ecdysterone:20-hydroxyecdysone.:Introduction and Its Benefit Applications.
  2.Beta-Ecdysterone and Its findings......
  3.Research update of Beta-Ecdysterone and Its findings.Cyanotis arachnoides Extract Related......

Claims & Warning:

Claims: Information this web site presented is meant for Nutritional Benefit and as an educational starting point only, for use in maintenance and promotion good health in cooperation with a common knowledge base reference...Furthermore,it based solely on the traditional and historic use or legend of a given herb from the garden of Adonis. Although every effort has been made to ensure its accurate, please note that some info may be outdated by more recent scientific developments......

Pharmakon Warning: The order of knowledge is not the transparent order of forms and ideas,as one might be tempted retrospectively to interpret it; it is the antidote....(Dissemination,Plato's Pharmacy,II.The Ingredients:Phantasms,Festivals,and Paints;138cf. Jacques Derrida.).

And as it happens,the technique of imitation,along with the production of the simulacrum,has always been in Plato's eyes manifestly magical,thaumaturgical:......and the same things appear bent and straight to those who view them in water and out,or concave and convex,owing to similar errors of vision about colors, and there is obviously every confusion of this sort in our souls.And so scene painting (skiagraphia) in its exploitation of this weakness of four nature falls nothing short of witchcraft (thaumatopoia), and so do jugglery and many other such contrivances.(Republic X,602c-d;cf.also 607c).