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Phytoecdysteroids (PEs) are naturally occurring steroid compounds, that have recently gained significant attention, due to their diverse biological activities and high therapeutic potential. The aim of the present study was to quantify some PEs including 20-hydroxyecdysterone (20-HE), ponasterone A (PA), and turkesterone (TU) in selected plant foods and Rhaponticum carthamoides extract. Furthermore, the effects of 20-HE, TU, and R. carthamoides extract, were investigated with in vitro methods using isolated smooth muscle tissues. The levels of 20-HE in the analyzed samples exhibited significant differences, with kaniwa seed extract containing the highest amount, followed by spinach leaf extract, quinoa seed extract, and asparagus stem extract. The in vitro analyses suggested that R. carthamoides extract exhibits dose-dependent cytotoxic effects on smooth muscle cells, with low doses promoting contraction and higher doses inducing relaxation. Additionally, the extract demonstrated a significant inhibitory effect on ACh-induced contractions, while 20-HE enhanced the contractile response. The current findings highlighted phytoecdysteroids' potential for modifying gastrointestinal motility.

The parasite Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that affects the lives of millions of indigenous people in Latin America. As medications to treat Chagas disease are limited to the application of benznidazole and nifurtimox, which are not ideal treatments for the chronic stage of the disease, the search for new antichagasic drug candidates is an important need. Ecdysone has previously been shown to interfere with the life cycle of T. cruzi . Here, we report the biological profiling and subsequent semisynthetic structure optimization of 47 ecdysteroids against T. cruzi with the aim of identifying selective trypanocidal ecdysteroids. Two moderately trypanocidal pharmacophores were identified: ecdysteroids containing a 6- tert -butyl oxime ether and a cinnamic ester moiety. These functional groups were combined into the structures of four new semisynthetic ecdysteroids ( 44 - 47 ), among which 44 exerted potent and selective trypanocidal activity (IC 50 < 2 μM). Cellular infection assays showed that ecdysteroid 44 potently and efficiently inhibited amastigote replication as determined by trypomastigote release after cellular infection with an IC 50 of 2.7 ± 0.1 μM. The compound was similarly potent to benznidazole (IC 50 = 3.8 ± 0.7 μM) and more than 5-fold more cytotoxic toward T. cruzi over RAW264.7 host macrophages. Overall, the ecdysteroid cinnamate ester 44 is a novel trypanocidal lead structure that needs to be further characterized in follow-up studies.

A comprehensive metabolite profiling of the medicinal plant Silene viridiflora using an UHPLC-ESI-MS/MS method is described for the first time. A total of 71 compounds were identified and annotated, the most common of which were flavonoids, triterpene glycosides, and ecdysteroids. The three major compounds schaftoside, 26-hydroxyecdysone, and silviridoside can be chosen as the markers for the assessment of the quality of S. viridiflora preparations. The methanol extract and a variety of metabolites identified in S. viridiflora were screened for their cytotoxic and Wnt pathway-inhibiting activities against triple-negative breast cancer (TNBC), the deadliest form of cancer in women. 2-Deoxy-20-hydroxyecdysone with submicromolar IC 50 was identified as a result. The structure-activity relationship derived from the data from the in vitro proliferation assay showed that the hydroxyl group present at position C-2 of steroid core reduces the ecdysteroids' cytotoxicity against cancer cells.

Diabetes is a significant global health challenge, affecting circa 540 million adults worldwide. Dioscorea dumetorum, a Nigerian folkloric antidiabetic plant is severely understudied in terms of its bioactive phytochemical constituents. Antidiabetic guided isolation of the tubers and peels of D. dumetorum afforded three phytoecdysteroids bearing a cis-fused A/B ring junction including two new ones: 24-hydroxymuristerone A (1) and 24-hydroxykaladasterone (2), alongside the known muristerone A (3). Additionally, 2,2',7,7'-tetramethoxy-[1,1'-biphenanthrene]-4,4',6,6'-tetraol (4), batatasin I (5), and dihydroresveratrol (6) were isolated. Structural elucidation relied on spectroscopic, spectrometric methods, and comparison with existing literature. The ethyl acetate extracts of both the tubers and peels of D. dumetorum exhibited the highest phenolic content, correlating with potent antioxidant activity. Compounds 4 (IC 50  = 0.10 mg/mL) and 6 (IC 50  = 0.22 mg/mL) demonstrated superior inhibitory effects against α-glucosidase compared to acarbose (IC 50  = 0.63 mg/mL). In contrast, compounds 3, 4, and 5 showed reduced α-amylase inhibition, with IC 50 values of 2.58, 3.78, and 1.12 mg/mL, respectively, compared to acarbose (IC 50  = 0.42 mg/mL). These observed bioactivities validate the traditional use of D. dumetorum and contribute valuable phytochemical data to the scientific literature of the species., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The roots of Fibraurea recisa are recognized as a rich source of protoberberine and aporphine alkaloids, but the non-alkaloidal metabolites in this plant are underexplored. The present study investigated the chemical composition of the plant roots using untargeted metabolomics-based molecular networking and MS2LDA motif annotation, revealing the presence of a characteristic fragment motif related to several sinapoyl-functionalized metabolites. Guided by the targeted motif, two new sinapic acid-ecdysteroid hybrids, named 3- O -sinapoyl makisterone A ( 1 ) and 2- O -sinapoyl makisterone A ( 2 ), were isolated. The structures of these compounds, including their absolute configuration, were elucidated by HR-ESIQTOFMS, MS 2 fragmentation, NMR spectroscopy, and chemical degradation coupled with optical rotation measurements. Although neither compound inhibited nitric oxide (NO) production or inducible nitric oxide synthase (iNOS) protein expression on lipopolysaccharide-induced RAW 264 cells, 2 significantly suppressed cyclooxygenase 2 (COX-2) protein expression at 1-30 μM. Additionally, decreased expression of COX-2 protein was barely observed after treatment with methyl sinapate or makisterone A, the steroid skeleton of 1 and 2 . These results indicated that the presence of the sinapoyl moiety at C-2 on the C 28 -ecdysteroid skeleton played a key role in the selectivity for the suppression of the COX-2 protein expression.

Six undescribed C 27 -phytoecdysteroid derivatives, named superecdysones A-F, and ten known analogs were extracted from the whole plant of Dianthus superbus L. Their structures were identified by extensive spectroscopy, mass spectrometric methods, chemical transformations, chiral HPLC analysis, and the single-crystal X-ray diffraction analysis. Superecdysones A and B possess a tetrahydrofuran ring in the side chain and superecdysones C-E are rare phytoecdysones containing a (R)-lactic acid moiety, whereas superecdysone F is an uncommon B-ring-modified ecdysone. Notably, based on the variable temperature (from 333 K to 253 K) NMR experiments of superecdysone C, the missing carbon signals were visible at 253 K and assigned. The neuroinflammatory bioassay of all compounds were evaluated, and 22-acetyl-2-deoxyecdysone, 2-deoxy-20-hydroxyecdysone, 20-hydroxyecdysone, ecdysterone-22-O-benzoate, 20-hydroxyecdysone-20,22-O-R-ethylidene, and acetonide derivative 20-hydroxyecdysterone-20, 22-acetonide significantly suppressed the LPS-induced nitric oxide generation in microglia cells (BV-2), with IC 50 values ranging from 6.9 to 23.0 μM. Structure-activity relationships were also discussed. Molecular docking simulations of the active compounds confirmed the possible mechanism of action against neuroinflammations. Furthermore, none compounds showed cytotoxicity against HepG2 and MCF-7. It is the first report about the occurrence and anti-neuroinflammatory activity of the phytoecdysteroids in the genus Dianthus. Our findings demonstrated that ecdysteroids may be used as potential anti-inflammatory drugs., Competing Interests: Declaration of competing interest The authors declare that the research was performed in the absence of any commercial or financial relationships that may be construed as a potential conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Ecdysteroid-containing herbal extracts, commonly prepared from the roots of Cyanotis arachnoidea , are marketed worldwide as a "green" anabolic food supplement. Herein are reported the isolation and complete 1 H and 13 C NMR signal assignments of three new minor ecdysteroids (compounds 2 - 4 ) from this extract. Compound 4 was identified as a possible artifact that gradually forms through the autoxidation of calonysterone. The compounds tested demonstrated a significant protective effect on the blood-brain barrier endothelial cells against oxidative stress or inflammation at a concentration of 1 μM. Based on these results, minor ecdysteroids present in food supplements may offer health benefits in various neurodegenerative disease states.

Glutathione S -transferases (GSTs) are conserved in a wide range of organisms, including insects. In 2014, an epsilon GST, known as Noppera-bo (Nobo), was shown to regulate the biosynthesis of ecdysteroid, the principal steroid hormone in insects. Studies on fruit flies, Drosophila melanogaster , and silkworms, Bombyx mori , demonstrated that loss-of-function mutants of nobo fail to synthesize ecdysteroid and die during development, consistent with the essential function of ecdysteroids in insect molting and metamorphosis. This genetic evidence suggests that chemical compounds that inhibit activity of Nobo could be insect growth regulators (IGRs) that kill insects by disrupting their molting and metamorphosis. In addition, because nobo is conserved only in Diptera and Lepidoptera, a Nobo inhibitor could be used to target IGRs in a narrow spectrum of insect taxa. Dipterans include mosquitoes, some of which are vectors of diseases such as malaria and dengue fever. Given that mosquito control is essential to reduce mosquito-borne diseases, new IGRs that specifically kill mosquito vectors are always in demand. We have addressed this issue by identifying and characterizing several chemical compounds that inhibit Nobo protein in both D. melanogaster and the yellow fever mosquito, Aedes aegypti . In this review, we summarize our findings from the search for Nobo inhibitors.

Owing to its high production and world-wide usage, plastic pollution is an increasing concern in marine environments. Plastic is decomposed into nano- and micro-sized debris, which negative affect reproduction and development in aquatic organisms. Bisphenol A (BPA), an additive of plastic, is released into the water column upon plastic degradation, and is known as a representative endocrine-disrupting chemical. However, the reproductive effects of plastics and bisphenols at the molecular level have not yet been explored in small marine crustaceans. In this study, we investigated the effects of polystyrene (PS) beads (0.05, 0.5, and 6 - μm) and bisphenol analogues (BPs; BPA, BPS, and BPF) on reproduction and development of small marine crustaceans. Effects on transcriptional changes in ecdysteroid and juvenile hormone (JH) signaling pathway-related genes were examined in the brackish water flea Diaphanosoma celebensis exposed to PS beads and BPs for 48 h. As results, BPs and PS beads delayed emergence time of first offspring, and increased fecundity in a concentration-dependent manner. BPs differentially modulated the expression of ecdysteroid and JH signaling pathway-related genes, indicating that BP analogs can disrupt endocrine systems via mechanisms different from those of BPA. PS beads was also changed the gene expression of both pathway, depending on their size and concentration. Our findings suggest that BP analogues and PS beads disrupt the endocrine system by modulating the hormonal pathways, affecting reproduction negatively. This study provides a better understanding of the molecular mode of action of BPs and PS beads in the reproduction of small crustaceans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Rice crab co-culture is a new integrated farming model in China. The application of triazole plant growth regulators (PRGs) is often used as an advantageous option to combat rice lodging. However, there is still a gap regarding the toxicity of these PRGs on the growth and development of the Chinese mitten crab (Eriocheir sinensis, E. sinensis). Here the effect of triazoles (paclobutrazol and uniconazole) on the molting mechanism of E. sinensis was investigated. Monitoring of regulatory genes associated with molting showed that the two PRGs were found to inhibit the expression of ecdysteroid hormone (EH), ecdysteroid receptors gene (EcR), and retinoid X receptors gene (RXR) and induce secretion of molt-inhibiting hormone (MIH) gene. In addition, the activities of chitinase (CHIA) and N-acetyl-β-d-aminoglucosidase (β-NAGase) were also inhibited by exposure to PRGs. Exposure to PRGs also elevated the mRNA expression of the growth-related myostatin gene (MSTN). These results revealed that there is a long-term risk of exposure to triazoles PRGs that may inhibit molting and affect normal development and immune system of E. sinensis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Six undescribed compounds, including four undescribed ecdysteroids (cyathsterones A-D) and two undescribed phenolic glycosides (cyathglucosides A-B), were isolated from the roots of Cyathula officinalis Kuan. Their structures were based on chemical analyses, NMR spectroscopic evidence, DP4+ calculations, and hydrolysis products. All compounds inhibited NO release in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages in vitro. Among them, cyathsterone A showed the strongest inhibitory effects. Moreover, cyathsterone A has been shown to inhibit the release of the proinflammatory cytokines TNF-α, IL-6, and IL-1β in LPS-induced RAW 264.7 macrophages in vitro. Further studies found that cyathsterone A present concentration-dependent suppression of the protein expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells in vitro and exerted anti-inflammatory activity via the NF-κB signalling pathway., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Two new ecdysteroids 14-epi-polypodine B (1) and 22-oxo-hydroxyecdysterone (2), along with nine known compounds, polypodine B (3), viticosterone E (4), 20-hdroxyecdysone-2-acetate (5), 22-oxo-20-hydroxyecdysone (6), 5-hydroxyecdysone (7), pinnatasterone (8), 3-epi-20-hydroxyecdysone (9), ecdysterone (10) and stachysterone B (11), were isolated from the aerial parts of Paris verticillata. The structures of all compounds were elucidated by extensive spectroscopic analysis, quantum chemical calculations and ANN-PRA/DP4 + probability analysis. Among them, the absolute configuration of compound 1 and 2 was unambiguous determined by ECD. Also, the isolated compounds were assessed for their cytotoxic activities. Compounds 2, 3 and 7 exhibited significant cytotoxic activities against PC12, LN299 and SMCC7721 cells., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.)

Gamma-ray radiation is a unique way to induce chemical transformations of bioactive compounds. In the present study, we pursued this approach to the diversity-oriented synthesis of analogs of 20-hydroxyecdysone (20E), an abundant ecdysteroid with a range of beneficial, non-hormonal bioactivities in mammals including humans. Gamma irradiations of aqueous solutions of 20E were conducted either in N 2 - or N 2 O-saturated solutions. Centrifugal partition chromatography was used to fractionate crude resulting irradiated materials using a biphasic solvent system composed of tert-butyl alcohol - ethyl acetate - water (0.45:0.9:1, v/v/v) in ascending mode. Subsequently, the products were purified by RP-HPLC. Fourteen ecdysteroids, including five new compounds, were isolated, and their structure were elucidated by 1D and 2D NMR and HRMS. Compounds 2-4, 7, 9, 12 and 15 were tested for their capacity to increase the Akt- and AMPK-phosphorylation of C2C12 murine skeletal myotubes in vitro. The compounds were similarly active on Akt as their parent compound. Stachysterone B (7) and a new ring-rearranged compound (12) were more potent than 20E in activating AMPK, indicating a stronger cytoprotective effect. Our results demonstrate the use of gamma irradiation in expanding the chemical diversity of ecdysteroids to obtain new, unusual bioactive metabolites., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Genetically uniform plant material, derived from Lychnis flos-cuculi propagated in vitro, was used for the isolation of 20-hydroxyecdysone and polypodine B and subjected to an evaluation of the antifungal and antiamoebic activity. The activity of 80% aqueous methanolic extracts, their fractions, and isolated ecdysteroids were studied against pathogenic Acanthamoeba castellani . Additionally, a Microtox ® acute toxicity assay was performed. It was found that an 80% methanolic fraction of root extract exerts the most potent amoebicidal activity at IC 50 of 0.06 mg/mL at the 3rd day of treatment. Both ecdysteroids show comparable activity at IC 50 of 0.07 mg/mL. The acute toxicity of 80% fractions at similar concentrations is significantly higher than that of 40% fractions. Crude extracts exhibited moderate antifungal activity, with a minimum inhibitory concentration (MIC) within the range of 1.25-2.5 mg/mL. To the best of our knowledge, the present report is the first to show the biological activity of L. flos-cuculi in terms of the antifungal and antiamoebic activities and acute toxicity. It is also the first isolation of the main ecdysteroids from L. flos-cuculi micropropagated, ecdysteroid-rich plant material.

The endocrine system of crustaceans regulates the molt cycle with ecdysteroid hormones, mainly the 20-hydroxyecdysone (20-HE). Moreover, the molt process requires the action of chitinolytic enzymes (e.g., chitinase, chitobiase) to break down the old cuticle. However, endocrine disrupting compounds (EDC) are capable of altering their normal functioning. Glyphosate-based herbicides (GBH), such as Roundup®, the most widely used herbicides, are found in freshwater environments and have been considered EDC for many aquatic organisms. Therefore, this study examined the effects of environmentally relevant GBH concentrations (0.0065, 0.065, and 0.28 mg L -1 ) on the 20-HE concentration and chitobiase activity in the decapod prawn Macrobrachium potiuna exposed for 14 days. Additionally, lipid peroxidation, a biomarker of membrane lipid degradation, was evaluated in hepatopancreas to assess cellular damage. Results showed that GBH decreased the 20-HE concentration in females at the two highest concentrations tested, while an increase was observed in males exposed to the highest GBH concentration. In addition, GBH also decreased chitobiase activity in males (all concentrations) and females (the two highest concentrations). Finally, GBH caused increased lipid peroxidation in males, indicating cellular damage in the hepatopancreas. In conclusion, this work suggests that GBH is an EDC for crustaceans by disrupting molting, which could lead to altered reproduction and thus population dynamics. Graphical abstract Decrease in the 20-HE concentration and chitobiase activity in muscle of males and females of the freshwater prawn Macrobrachium potiuna exposed to the herbicide Roundup® for 14 days.

Molting enables growth and development across ecdysozoa. The molting process is strictly controlled by hormones - ecdysteroids. Ecdysteroidogenesis occurs in theprothoracic glands and stimulated by prothoracicotropic hormone in insects, while it ensues in the Y-organ and regulated by the molt inhibiting hormone in crustaceans. A peak in ecdysteroids in the hemolymph induces a cascade of multiple neuropeptides including Ecdysis Triggering Hormone (ETH) and Corazonin. The role of ETH is well defined in controlling the molt process in insects, but it is yet to be defined in crustaceans. In this study, we investigated the behavioral response of intermolt crayfish to ETH and Corazonin injections as well as the impact of ETH on the molt period using in vivo assays. Injection of Corazonin and ETH resulted in a clear and immediate eye twitching response to these two neuropeptides. The Corazonin injection induced eye twitching in slow and asynchronous manner, while ETH injection caused eye twitching in a relatively fast and synchronous way. A single injection of ETH to crayfish resulted in a remarkable prolong molt period, at twice the normal molting cycle, suggesting that ETH plays a key role in controlling the molt cycle in decapod crustaceans. Given the key significance of ETH in molt regulation and its plausible application in pest control, we characterized ETH across the pancrustacean orders. Bioinformatic analysis shows the mature ETH sequence is identical in all studied decapod species. ETH can be classified into specific groups based on the associated motif in each insect order and shows an insect motif -KxxPRx to be conserved in crustaceans., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Steroid hormones are crucial regulators of life-stage transitions during development in animals. However, the molecular mechanisms by which developmental transition through these stages is coupled with optimal metabolic homeostasis remains poorly understood. Here, we demonstrate through mathematical modelling and experimental validation that ecdysteroid-induced metabolic remodelling from resource consumption to conservation can be a successful life-history strategy to maximize fitness in Drosophila larvae in a fluctuating environment. Specifically, the ecdysteroid-inducible protein ImpL2 protects against hydrolysis of circulating trehalose following pupal commitment in larvae. Stored glycogen and triglycerides in the fat body are also conserved, even under fasting conditions. Moreover, pupal commitment dictates reduced energy expenditure upon starvation to maintain available resources, thus negotiating trade-offs in resource allocation at the physiological and behavioural levels. The optimal stage-specific metabolic shift elucidated by our predictive and empirical approaches reveals that Drosophila has developed a highly controlled system for ensuring robust development that may be conserved among higher-order organisms in response to intrinsic and extrinsic cues.

The expression of multidrug resistance P-glycoprotein (P-gp) by cancer cells represents one of the major drawbacks to successful cancer therapy. Accordingly, the development of drugs that inhibit the activity of this transporter remains a major challenge in cancer drug discovery. In this context, several new ecdysteroid derivatives have been synthesized and evaluated as P-gp inhibitors. Two of them (compounds 9 and 14 ) were able to resensitize CEM Vbl100 and LoVo Doxo resistant cell lines to vinblastine and doxorubicin, respectively. Indeed, both compounds 9 and 14 increased the cellular accumulation of rhodamine 123 in cells expressing P-gp and stimulated basal P-glycoprotein-ATPase activity at a 1 μM concentration, demonstrating their interference with the transport of other substrates in a competitive mode. Moreover, in a medulloblastoma cell line (DAOY), compounds 9 and 14 reduced the side population representing cancer stem cells, which are characterized by a high expression of ABC drug transporters. Further, in DAOY cells, the same two compounds synergized with cisplatin and vincristine, two drugs used commonly in the therapy of medulloblastoma. Molecular docking studies on the homology-modeled structure of the human P-glycoprotein provided a rationale for the biological results, validating the binding mode within the receptor site, in accordance with lipophilicity data and observed structure-activity relationship information. Altogether, the present results endorse these derivatives as promising P-gp inhibitors, and they may serve as candidates to reverse drug resistance in cancer cells.

Sphenocentrum jollyanum seeds (MeOH extract and n butanol fraction) exhibited urease inhibitory activity (IC 50 40.0 ± 0.92, 28.6 ± 0.41). The Ethyl acetate (EtOAc) fraction gave significant antacid activity with an increase in the baseline pH value of 1.2 to 1.61 ± 0.00 and 1.53 ± 0.00 at 50 and 100 mg, respectively, compared to the antacid activity of sodium bicarbonate (1.53 ± 0.00, 1.47 ± 0.00). Five known ecdysteroid compounds isolated from S. jollyanum ethyl acetate and n butanol fractions are Pinnatasterone (1), Polypodine B (2), 20-hydroxyecdysone (3), 20, 26-dihydroxyecdysone, (4) and Atrotosterone A (5). The compounds' structures were determined using extensive 1D and 2D NMR experiments, and the molecular mass for each of the compounds was confirmed by FAB-MS. Compounds 1-5 were evaluated for their urease inhibitory and antacid activities. Fractions were active in comparison with the standard drug acetohydroxamic acid, and sodium bicarbonate, respectively. Compounds 2, 3 and 1 showed significant urease inhibitory activity (IC 50 7.0 ± 0.56, 13.8 ± 0.49 and 14.1 ± 0.59), respectively. The activity of compounds 4 and 5 were moderate compared to that of acetohydroxamic acid (IC 50 value 20.3 ± 0.43). Very few compounds have been isolated from this plant despite the numerous biological activities reported for it. The antacid and urease inhibitory activities of this plant and isolated compounds are described for the first time., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ecdysteroids are widely found in terrestrial organisms, including insects, crustaceans, fungi, and plants. The function of ecdysteroids has been extensively studied in insects for decades because ecdysteroids regulate metamorphosis. In plants, in contrast, ecdysteroids (called phytoecdysteroids) do not show apparent hormonal activity and their function remains unclear. However, it has been proposed that phytoecdysteroids have an antifeedant function. Ecdysteroid ingestion disrupts insect development and alters behavior to deter insect feeding, resulting in reduced plant damage by the insect. These points of view are generally accepted, but the function of phytoecdysteroids in specific contexts has not been unveiled. In the present study, we used larvae of the silkworm, Bombyx mori, to investigate the biological significance of phytoecdysteroids. To mimic the situation where larvae consume plant leaves that contain phytoecdysteroids, 26 or 30 larvae were fed the diet containing ecdysteroid or the control diet. We show that ecdysteroid ingestion dramatically suppressed carbohydrate processing in the larval midgut to reduce the nutritional value of the ingested diet. Based on the present results, we propose a new explanation of phytoecdysteroid function: ingested ecdysteroids may lead to the erroneous perception that the plant is poor in nutrients and consequently result in cessation of feeding.

This article presents the results of comprehensive studies to analyze the effect of a mixture of phytoecdysteroids extracted from the juice of Serratula coronata L. on the productivity and vitality of ducklings when grown for meat, and the optimal doses of its inclusion in the diet of the bird are revealed. The methodological basis of this study was the earlier works of domestic and foreign scientists on the topic under study. In the studies, a mixture of ecdysteroids extracted from the juice of the Serratula coronata L. was used according to the method developed by a team of scientists of the Ufa Federal Research Center of the Russian Academy of Sciences (Patent RU 2151598). The object of the study was the young ducks of the cross breed "Agidel 34" of the Beijing breed. It was established that the use of phytoecdysteroids in the diets of ducklings at a dose of 1.0 mg/l of drinking water allowed to increase the safety of the livestock by 4.0%, live weight by 4.5% (p <  0.01), average daily live weight gain by 3.0-3.5%, gutted carcass weight - 7.1%. At the same time, feed costs per unit of production decreased by 2.0%, and the profitability of duck meat production increased by 5.2%., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

To characterize the potential endocrine-disrupting chemicals (EDCs) in the environment that interact with the crustacean ecdysone receptor (EcR), we established a method involving in silico modeling/molecular docking and in vitro reporter gene assay. Cherry shrimp (Neocaridina davidi) EcR (NdEcR) and retinoid X receptor (NdRxR) were identified and cloned for use in this method. A theoretical 3D model of NdEcR ligand-binding domain (LBD) was built in silico based on sequence homology with the established X-ray structure of insect EcR. The interaction of the NdEcR LBD with ecdysteroids, diacylhydrazine (DAH) pesticides, and other potential EDCs was evaluated using molecular docking programs. The results revealed that the ligand-binding pocket in the NdEcR LBD was flexible and adaptive for accommodating ligands of different shapes. The agonistic and antagonistic activities of the candidate compounds were further assessed by in vitro reporter gene assay using human cell lines transiently transfected with NdEcR and NdRxR expression plasmids and a reporter plasmid containing synthesized ecdysone response element. The assay was validated by the dose-dependent responses of EcR-mediated gene transcription after treating the transfected cell lines with ecdysteroids, 20-hydroxyecdysone, and ponasterone A. Examination of the candidate compounds using the reporter gene assay revealed restricted functional specificity to ecdysteroids and DAHs. Three of the tested DAH pesticides originally targeting the insect EcR were found to be weak agonists and strong antagonists of NdEcR. These results suggest that DAHs are potential EDCs for crustaceans that disrupt their ecdysteroid signals by functioning as EcR agonists or antagonists., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Size polymorphism is common in bees, and is determined by environmental factors such as temperature, brood cell size, and the diet provided to developing larvae. In social bees, these factors are further influenced by intricate interactions between the queen, workers, and the developing brood which eventually determine the final size and caste of developing larvae. Environmental and social factors act in part on juvenile hormone and ecdysteroids, which are key hormonal regulators of body size and caste determination. In some social bees, body size variation is central for social organization because it structures reproductive division of labor, task allocation among workers, or both. At ecological scales, body size also impacts bee-mediated pollination services in solitary and social species by influencing floral visitation and pollination efficacy., (Copyright © 2019. Published by Elsevier Inc.)

In the present study, the roles of a major serine/threonine protein phosphatase 2A (PP2A) in prothoracicotropic hormone (PTTH)-stimulated prothoracic glands (PGs) of Bombyx mori were evaluated. Immunoblotting analysis showed that Bombyx PGs contained a structural A subunit (A), a regulatory B subunit (B), and a catalytic C subunit (C), with each subunit undergoing development-specific changes. The protein levels of each subunit were not affected by PTTH treatment. However, the highly conserved tyrosine dephosphorylation of PP2A C subunit (PP2Ac), which appears to be related to activity, was increased by PTTH treatment in a time-dependent manner. We further demonstrated that phospholipase C (PLC), Ca 2+ , and reactive oxygen species (ROS) are upstream signaling for the PTTH-stimulated dephosphorylation of PP2Ac. The determination of PP2A enzymatic activity showed that PP2A enzymatic activity was stimulated by PTTH treatment both in vitro and in vivo. Okadaic acid (OA), a specific PP2A inhibitor, prevented the PTTH-stimulated dephosphorylation of PP2Ac and reduced both basal and PTTH-stimulated PP2A enzymatic activity. The determination of ecdysteroid secretion showed that treatment with OA did not affect basal ecdysteroid secretion but did significantly inhibit PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated PP2A activity is involved in ecdysteroidogenesis. Treatment with OA stimulated the basal phosphorylation of the extracellular signal-regulated kinase (ERK) and 4E-binding protein (4E-BP) without affecting PTTH-stimulated ERK and 4E-BP phosphorylation. From these results, we hypothesize that PTTH-regulated PP2A signaling is a necessary component for the stimulation of ecdysteroidogenesis, potentially by mediating the link between ERK and TOR signaling pathways., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Phytoecdysteroids exert their non-hormonal anabolic and adaptogenic effects in mammals, including humans, through a partially revealed mechanism of action involving the activation of protein kinase B (Akt). We have recently found that poststerone, a side-chain cleaved in vivo metabolite of 20-hydroxyecdysone, exerts potent anabolic activity in rats. Here we report the semi-synthetic preparation of a series of side-chain cleaved ecdysteroids and their activity on the Akt phosphorylation in murine skeletal muscle cells. Twelve C-21 ecdysteroids including 8 new compounds were obtained through the oxidative side-chain cleavage of various phytoecdysteroids, or through the base-catalyzed autoxidation of poststerone. The complete 1 H and 13 C NMR spectroscopic assignments of the new compounds are presented. Among the tested compounds, 9 could activate Akt stronger than poststerone revealing that side-chain cleaved derivatives of phytoecdysteroids other than 20-hydroxyecdysone are valuable bioactive metabolites. Thus, our results suggest that the expectable in vivo formation of such compounds should contribute to the bioactivity of herbal preparations containing ecdysteroid mixtures., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Despite a large occurrence, especially over the Pacific Ocean, the chemical diversity of marine invertebrates belonging to the order Zoantharia is largely underexplored. For the two species of the genus Antipathozoanthus no chemical study has been reported so far. The first chemical investigation of Antipathozoanthus hickmani collected at the Marine Protected Area "El Pelado", Santa Elena, Ecuador, led to the isolation of four new ecdysteroid derivatives named ecdysonelactones. The structures of ecdysonelactones A-D ( 1 - 4 ) were determined based on their spectroscopy data, including 1D and 2D NMR and HRMS. The four compounds of this family of ecdysteroids feature an unprecedented γ-lactone fused at the C-2/C-3 position of ring A. These derivatives exhibited neither antimicrobial nor cytotoxic activities.

Multidrug resistance is a widespread problem among various diseases and cancer is no exception. We had previously described the chemo-sensitizing activity of ecdysteroid derivatives with low polarity on drug susceptible and multi-drug resistant (MDR) cancer cells. We have also shown that these molecules have a marked selectivity towards the MDR cells. Recent studies on the oximation of various steroid derivatives indicated remarkable increase in their antitumor activity, but there is no related bioactivity data on ecdysteroid oximes. In our present study, 13 novel ecdysteroid derivatives (oximes, oxime ethers and a lactam) and one known compound were synthesized from 20-hydroxyecdysone 2,3;20,22-diacetonide and fully characterized by comprehensive NMR techniques revealing their complete 1 H and 13 C signal assignments. The compounds exerted moderate to strong in vitro antiproliferative activity on HeLa, SiHa, MCF-7 and MDA-MB-231 cell lines. Oxime and particularly oxime ether formation strongly increased their inhibitory activity on the efflux of rhodamine 123 by P-glycoprotein (P-gp), while the new ecdysteroid lactam did not interfere with the efflux function. All compounds exerted potent chemo-sensitizing activity towards doxorubicin on a mouse lymphoma cell line and on its MDR counterpart, and, on the latter, the lactam was found the most active. Because of its MDR-selective chemo-sensitizing activity with no functional effect on P-gp, this lactam is of high potential interest as a new lead for further antitumor studies., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

The present study investigated the protective effect of phytoecdysteroids extracted from the Ajuga iva plant on body weight changes, blood glucose, insulin total protein, blood urea nitrogen (BUN), creatinine, triglycerides (TG), cholesterol, lipid peroxidation, antioxidant enzymes, pancreatic histopathology and hexokinase-I expression in the alloxan-induced diabetic rats. Experimental diabetes was induced following 15day intraperitoneal administration of alloxan. The rats were divided into four groups. Group I served as a sham group, and group II served as the diabetic control. Group III served as a treatment for phytoecdysteroids (10mg/kg), and group IV served as a treatment for phytoecdysteroids (20mg/kg). Phytoecdysteroids restored body weight loss to its antihyperglycemic effect. Blood glucose was reduced 19.2 and 52.9% in group III and IV respectively. Blood insulin (54.9 and 105.88%) and total protein (25 and 72.2%) was increased in group III and IV respectively. BUN, creatinine, TG, cholesterol and lipid peroxidation was significantly reduced following treatment. Catalase, superoxide dismutase (SOD), and glutathione peroxidase activity were significantly increased following treatment. Islet β-cells are lost in alloxan-induced diabetic rats. Regeneration of islets and reduced atrophy of acinar cells were noted. The number of insulin-secreting cells was tremendously reduced in alloxan-induced diabetic rats. Insulin-secreting cells were increased 48 and 61% in group III and IV respectively. Hexokinase-I mRNA (28.3 & 93.5%) and protein (27.9 and 55.3%) expression were significantly increased following treatment. Taking all these data together, it is suggested that the phytoecdysteroid could be a potential therapeutic agent against experimental diabetes., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

High-speed counter-current chromatography was used to separate and purify ecdysteroids for the first time from the stems of Diploclisia glaucescens using a two-phase solvent system composed of ethyl acetate- n -butanol-ethanol-water (3:0.2:0.8:3, v / v ). Three ecdysteroids were obtained from 260 mg of ethyl acetate extract of the residue obtained after evaporation of the crude ethanolicextractof D. glaucescens in one-step separation, which were identified as paristerone ( I , 30.5 mg), ecdysterone ( II , 7.2 mg), and capitasterone ( III , 8.1 mg) by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). Their anti-inflammatory activities were evaluated by measuring the inhibitory ratios of β-glucuronidase release in rat polymorphonuclear leukocytes (PMNs) induced by platelet-activating factor. Compounds I - III showed significant anti-inflammatory activities with IC 50 -values ranging from 1.51 to 11.68 μM, respectively., Competing Interests: The authors declare no conflict of interest.

A new phytoecdysteroid compound, named Niuxixinsterone D (1), together with two known phytoecdysteroid compounds (2 and 3) were isolated from Achyranthes bidentata Bl.. The structure of the new compound was elucidated by extensive spectral analysis, including HR-ESI-MS, 1D and 2D NMR methods. Compounds 1-3 were tested for their inhibitory effects against LPS-induced NO production in RAW 264.7 macrophages, and compound 1 and 3 exhibited anti-neuroinflammatory activity with inhibited 29.7 and 26.0% NO production.

The eclosion gate in insect development is controlled by the circadian clock and hormonal cascade. To study mechanisms underlying the eclosion gate, we examined eclosion-timing signals from the circadian clock, and the role of 20-hydroxyecdysone in the eclosion gate of the flesh fly, Sarcophaga crassipalpis. Phase responses of the eclosion rhythm were examined by applying a low-temperature pulse in the day prior to the first eclosion peak. A low-temperature pulse applied about 5.4 h before eclosion advanced an eclosion peak by 0.9 h. This indicates that an interval from the Zeitgeber (external environmental cues) input to the behavioral output by the circadian clock is 4.5 h. Signals released by the circadian clock in the last 4.5 h before eclosion could change eclosion time. In the prothoracic gland, daily changes in immunoreactivity against a circadian clock protein PERIOD were observed in the last two days before eclosion. Hemolymph titers of 20-hydroxyecdysone were very low in the last two days of the pupal period. 20-hydroxyecdysone injections caused a delay, not an advancement, in eclosion time in a time dependent manner: pharate adults were sensitive to 20-hydroxyecdysone about 20 and 16 h before eclosion, whereas no significant effects were observed about 12 and 8 h before eclosion. These results suggest that 20-hydroxyecdysone is not a timing signal submitted by the circadian clock but an indicator to suppress premature eclosion. The circadian clock in the prothoracic gland presumably sends a signal distinct from ecdysteroids from several hours before eclosion to time the onset of eclosion.

The marine red algae of the genus Laurencia have been widely studied for their structurally diverse and biologically active secondary metabolites. We report here the natural product investigation of the organic extract of a newly identified South African endemic species, Laurencia alfredensis . A sequence of column chromatography, preparative TLC and normal phase HPLC resulted in the isolation of eleven compounds comprising three labdane-type diterpenes ( 1 - 3 ), four polyether triterpenes ( 4 - 7 ), three cholestane-type ecdysteroids ( 8 - 1 0 ) and a glycolipid ( 11 ). Compounds 1 - 3 , 5 - 8 and 10 have not previously been reported, while compound 9 is reported here for the first time from a natural source and the known compound 11 isolated for the first time from the genus Laurencia . The structural elucidation and the relative configuration assignments of the compounds were accomplished by extensive use of 1D- and 2D-NMR, HR-ESI-MS, UV and IR spectroscopic techniques, while the absolute configuration of compound 1 was determined by single-crystal X-ray diffraction analysis. All compounds were evaluated against the MDA-MB-231 breast and HeLa cervical cancer cell lines. Compound 2 exhibited low micromolar antiproliferative activity (IC 50 = 9.3 µM) against the triple negative breast carcinoma and compound 7 was similarly active (IC 50 = 8.8 µM) against the cervical cancer cell line.

A number of phytoecdysteroid compounds, such as ecdysterone, polipodin V, 2-deoxy-20-hydroxyecdysone, integristeron A and 2-deoxydizon were isolated from Stachys hissarica plant and their structures were confirmed by NMR, mass and IR spectroscopy. In addition, the biological activity of the S. hissarica plant's extract was tested on rats for wound healing activity. It was shown that the extract at repeated oral (per os) administration at a dose of 10 mg/kg speeds up the healing process of linear skin wounds in rats. The wound-healing activity of S. hissarica extract is confirmed to be effective and exceeds known drug methyluracil (2,4-dioxo-6-methyl-1,2,3,4-tetrahydropyrimidine), especially in case of alloxan induced diabetic animals.

A new marine ecdysteroid with an α-hydroxy group attaching at C-4 instead of attaching at C-2 and C-3, named palythone A (1), together with eight known compounds (2-9) were obtained from the ethanolic extract of the Formosan zoanthid Palythoa mutuki. The structures of those compounds were mainly determined by NMR spectroscopic data analyses. The absolute configuration of 1 was further confirmed by comparing experimental and calculated circular dichroism (CD) spectra. Anti-dengue virus 2 activity and cytotoxicity of five isolated compounds were evaluated using virus infectious system and [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assays, respectively. As a result, peridinin (9) exhibited strong antiviral activity (IC50 = 4.50 ± 0.46 μg/mL), which is better than that of the positive control, 2'CMC. It is the first carotene-like substance possessing anti-dengue virus activity. In addition, the structural diversity and bioactivity of the isolates were compared by using a ChemGPS-NP computational analysis. The ChemGPS-NP data suggested natural products with anti-dengue virus activity locate closely in the chemical space.

The objective of this study was to evaluate the effects of supplementation of phytoecdysteroids (PEDS) extracted from Cyanotis arachnoidea on rumen fermentation, enzymes activity and microbial efficiency in a dual flow continuous-culture system. A single-factor experimental design was used with twelve fermenters in 4 groups with 3 replicates each. Fermenters were incubated for a total of 7 days that included first 4 days for adaptation and last 3 days for sampling. PEDS was added at levels of zero (as control), 5, 10, and 15 mg/g of the substrate (DM). The results showed that increasing supplementation levels of PEDS resulted in incremental digestibility of dry matter (DMD) (quadratic, P = 0.001) and organic matter (OMD) (quadratic, P = 0.031), but unchanged digestibility of neutral detergent fiber (NDFD), crude protein (CPD) and acid detergent acid (ADFD). As supplementation levels of PEDS increased, there were decreased response in the concentration of ammonia nitrogen (NH3-N) (linear, P = 0.015) and increased response in molar proportions of butyrate (linear, P = 0.004), but unchanged response in total volatile fatty acid (TVFA) and the molar proportion of acetate and propionate, respectively. Increasing PEDS supplementation levels decreased the ratio of acetate to propionate (linear, P = 0.038), suggesting an alteration of rumen fermentation pattern occurring due to PEDS supplementation in the diet. Supplementation of PEDS significantly increased activities of glutamate dehydrogenase (quadratic, P = 0.001), alanine dehydrogenase (quadratic, P = 0.004), glutamate synthase (linear, P = 0.038), glutamine synthetase (quadratic, P = 0.011), respectively. There were no discernible differences in the activity of carboxymethyl cellulose (CMCase), xylanase and protease regardless of the treatments. The daily production of microbial nitrogen (linear, P = 0.002) and microbial efficiency (MOEEF) (linear, P = 0.001) increased linearly as supplementation levels of PEDS increased. The decreased response of fluid NH3-N and the increased response of MN indicated that PEDS positively increased the synthesis of microbial proteins.

Sida tuberculata is found in a region of South America and has traditionally been consumed as an infusion or tea. The chemical composition and antifungal activity of aqueous infusions from leaves and roots were investigated. LC-ESI-MS mass spectra were successfully obtained and used to identify four ecdysteroids: 20-hydroxyecdysone-3-O-β-D-glycopyranoside, 20-hydroxyecdysone, 20-hydroxyecdysone-3-O-β-D-xylose and a hydroxyecdysterone derivative. The in vitro antifungal activity was studied, and the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) were established against Candida krusei isolates. The antibiofilm activity was evaluated by the determination of the biofilm removal efficiency in contaminated central venous catheter (CVC) coupons. The preparations exhibited antifungal activity against the species tested, with MICs ranging from 3.90 to 62.50 μg/ml. The infusion removed the C. krusei biofilm after 90 min of exposure. The observed bioactivity and composition of ecdysteroids will contribute to the future development of antifungal substances for clinical use or as food additives., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Arthropod molt is coordinated through the interplay between ecdysteroids and neuropeptide hormones. In crustaceans, changes in the activity of Y-organs during the molt cycle have been regulated by molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). Little has been known of the mode of direct effects of ecdysteroids on the levels of MIH and CHH in the eyestalk ganglia during the molt cycle. This study focused on a putative feedback of ecdysteroids on the expression levels of MIH transcripts using in vitro incubation study with ecdysteroids and in vivo RNAi in the blue crab, Callinectes sapidus. Our results show a specific expression of ecdysone receptor (EcR) in which EcR1 is the major isoform in eyestalk ganglia. The initial elevation of MIH expression at the early premolt stages is replicated by in vitro incubations of eyestalk ganglia with ecdysteroids that mimic the intrinsic conditions of D0 stage: the concentration (75 ng/ml) and composition (ponasterone A and 20-hydroxyecdysone at a 3:1 (w:w) ratio). Additionally, multiple injections of EcR1-dsRNA reduce MIH expression by 67%, compared to the controls. Our data provide evidence on a putative feedback mechanism of hormonal regulation during molting cycle, specifically how the molt cycle is repeated during the life cycle of crustaceans. The elevated concentrations of ecdysteroids at early premolt stage may act positively on the levels of MIH expression in the eyestalk ganglia. Subsequently, the increased MIH titers in the hemolymph at postmolt would inhibit the synthesis and release of ecdysteroids by Y-organs, resulting in re-setting the subsequent molt cycle.

A smad nuclear interacting protein 1 (SNIP1) homolog was identified in the crayfish Procambarus clarkii and this gene encoded a polypeptide of 288 amino acids. Phylogenic analysis showed that P. clarkii SNIP1 was highly homologous to that of invertebrates and shared a highly conserved FHA domain of SNIP proteins at the C-terminus. Quantitative real-time PCR (qRT-PCR) analysis showed that Pc-SNIP1 expression was higher in heart than that in other examined tissues. In addition, prokaryotic expression and purification of the recombinant SNIP1 protein were performed. SDS-PAGE and western blot analysis demonstrated that a 35 KDa recombinant protein was successfully expressed in Escherichia coli cells. The expression of Pc-SNIP1 was significantly up-regulated in hepatopancreas after 20-hydroxyecdysone induction. Knockdown of Pc-SNIP1 gene by small interfering RNA (siRNA) transfection had significant influence on the expression of some ecdysteroid-responsive genes in hepatopancreas, which were confirmed by qRT-PCR and western blot. All together, these results suggest that SNIP1 is involved in the physiological process regulated by ecdysteroid in P. clarkii., (© 2015 Wiley Periodicals, Inc.)

Herbs, herbal extracts, or phytochemicals are broadly used as foods, drugs, and as traditional medicines. These are well regulated in Europe, with thorough controls on both safety and efficacy or validity of health claims. However, the distinction between medicines and foods with health claims is not always clear. In addition, there are several cases of herbal products that claim benefits that are not scientifically demonstrated. This review details the European Union (EU) legislative framework that regulates the approval and marketing of herbal products bearing health claims as well as the scientific evidence that is needed to support such claims. To illustrate the latter, we focus on phytoecdysteroid (PE)-containing preparations, generally sold to sportsmen and bodybuilders. We review the limited published scientific evidence that supports claims for these products in humans. In addition, we model the in silico binding between different PEs and human nuclear receptors and discuss the implications of these putative bindings in terms of the mechanism of action of this family of compounds. We call for additional research to validate the safety and health-promoting properties of PEs and other herbal compounds, for the benefit of all consumers.

Microsorum grossum (Polypodiaceae), locally called metuapua'a, is one of the most frequently used fern species in Polynesian traditional medicine. Fronds or rhizomes of this species are common ingredients of popular medicine recipes to cure various ailments. M. grossum frond and rhizome extracts contain, as their main bioactive components, phytoecdysteroids such as 20-hydroxyecdysone, known to have many interesting biological activities and considered to be adaptogenic compounds [1]. The skin-active effect of M. grossum extract was investigated in two ways on human dermal fibroblasts: a transcriptomic study with c-DNA array for gene expression modulation and a Stress Induced Premature Senescence (SIPS) test. The total extract of M. grossum up-regulates Heme Oxygenase 1 (HO1), an enzyme which protects cells from oxidative stress and which is responsible for skin photoimmunoprotection. The present paper also reports that premature senescence of human skin induced by repeated UV irradiations can be prevented by an ecdysteroid fraction of M. grossum. Our data indicate that extracts of M. grossum could protect skin against oxidative stresses and suggest that they could be used as innovative active cosmetic ingredients.

Ecdysteroids, analogs of the insect molting hormone, are known for their various mild, nonhormonal bioactivities in mammals. Previously, we reported that less-polar ecdysteroids can modulate the doxorubicin resistance of a multidrug resistant (MDR) mouse lymphoma cell line expressing the human ABCB1 transporter. Here, we describe the ability of 20-hydroxyecdysone (1) and its mono- (2) and diacetonide (3) derivatives to sensitize various MDR and non-MDR cancer cell lines towards doxorubicin, paclitaxel, vincristine, or cisplatin. Drug IC50 values with or without ecdysteroid were determined by MTT assay. Compound 3 significantly sensitized all cell lines to each chemotherapeutic except for cisplatin, whose activity was decreased. In order to overcome solubility and stability issues for the future in vivo administration of compound 3, liposomal formulations were developed. By means of their combination index values obtained via checkerboard microplate method, a formulation showed superior activity to that of compound 3 alone. Because ecdysteroids act also on non-ABCB1 expressing (sensitive) cell lines, our results demonstrate that they do not or not exclusively exert their adjuvant anticancer activity as ABCB1 inhibitors, but other mechanisms must be involved, and they opened the way towards their in vivo bioactivity testing against various cancer xenografts.

RXR cDNA cloning from three Uca species led to the identification of 4 conserved isoforms, indicative of alternative splicing in the hinge and ligand binding domains (LBD). Sequencing of overlapping clones from a Ucapugilator genomic library identified EcR isoforms matching previously identified cDNA variants; in addition, a cryptic exon in the LBD was detected and evidence for expression of this new isoform was obtained from next-generation sequencing. RNA-seq analysis also identified a new amino terminal EcR variant. EcR and RXR transcript abundance increases throughout ovarian maturation in U. pugilator, while cognate receptor transcript abundance remains constant in a related Indo-Pacific species with a different reproductive strategy. To examine if crab RXR LBD isoforms have different physical properties in vitro, electromobility shift assays were performed with different EcR isoforms. The cognate crab and fruit fly receptors differ in their responses to hormone. Ecdysteroids did not increase DNA binding for the crab heterodimers, while ecdysteroids stimulate binding for Drosophilamelanogaster EcR/USP heterodimers. In swapping experiments, UpEcR/USP heterodimers did not show ligand-responsive differences in DNA binding; both crab RXR LBD isoforms, however, conferred ligand-responsive increases in DNA binding with DmEcRs. These data indicate that both UpRXR LBD isoforms can heterodimerize with the heterologous DmEcR receptors and promote ligand and DNA binding. Unresponsiveness of the cognate receptors to ecdysteroid, however, suggest additional factors may be required to mediate endogenous, perhaps isoform-specific, differences in EcR conformation, consistent with previously reported effects of UpRXR isoforms on UpEcR ligand-binding affinities., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The conditional expression of alternative life strategies is a widespread feature of animal life and a pivotal adaptation to life in seasonal environments. To optimally match suites of traits to seasonally changing ecological opportunities, animals living in seasonal environments need mechanisms linking information on environmental quality to resource allocation decisions. The butterfly Bicyclus anynana expresses alternative adult life histories in the alternating wet and dry seasons of its habitat as endpoints of divergent developmental pathways triggered by seasonal variation in preadult temperature. Pupal ecdysteroid hormone titers are correlated with the seasonal environment, but whether they play a functional role in coordinating the coupling of adult traits in the alternative life histories is unknown. Here, we show that manipulating pupal ecdysteroid levels is sufficient to mimic in direction and magnitude the shifts in adult reproductive resource allocation normally induced by seasonal temperature. Crucially, this allocation shift is accompanied by changes in ecologically relevant traits, including timing of reproduction, life span, and starvation resistance. Together, our results support a functional role for ecdysteroids during development in mediating strategic reproductive investment decisions in response to predictive indicators of environmental quality. This study provides a physiological mechanism for adaptive developmental plasticity, allowing organisms to cope with variable environments.

The univoltine Bactrocera minax (Enderlein) is a devastating pest of citrus in Nepal, India, Bhutan, and China. To better understand the overwintering pupal diapause, we investigated the juvenile hormone III (JH III) and ecdysteroid 20-hydroxyecdysone (20E) titers across the developmental stages, and the effects of juvenile hormone analog (JHA) and 20E treatments on adult emergence patterns. The results showed that both JH III and 20E levels fluctuated from the late larval stage to the late pupal stage. JHA and 20E treatments at the late larval stage had marginal effect on the adult emergence patterns, with slightly faster adult eclosion and higher eclosion rate. Similarly, JHA treatment at the early pupal stage did not affect the adult emergence. However, 20E treatment at the early pupal stage remarkably shortened the duration of diapause, but lowered the eclosion rate, especially with relatively high dose. These findings demonstrated that 20E treatment at the early pupal stage is a suitable method to hasten the diapause termination of B. minax, which will contribute to the rearing of B. minax in the laboratory as needed for routine experiments.

Steroid hormones classically mediate their actions by binding to intracellular receptor proteins that migrate to the nucleus and act as transcription factors to change gene expression. However, evidence is now accumulating for rapid, non-genomic effects of steroids. There is considerable controversy over the mechanisms underlying such effects. In a number of cases evidence has been presented for the direct activation of G-protein coupled receptors (GPCRs) by steroids, either at the plasma membrane, or at intracellular locations. Here, we will focus on the non-genomic actions of ecdysteroids on a Drosophila GPCR, DopEcR (CG18314), which can be activated by both ecdysone and the catecholamine, dopamine. We will also point out parallels between this system and the activation of the vertebrate GPCR, GPER1 (GPR30), which is thought to be activated by 17β-estradiol. We propose that the cellular localization and signalling properties of both DopEcR and GPER1 may be cell specific and depend upon their interactions with both accessory molecules and signalling pathways., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

The impact of the 15-day consumption of Serratula coronata extract containing phytoecdysteroids on some indicators of hormonal status and activity of apoptosis in various organs of growing male Wistar rats (initial body weight 127.8 +/-2.5 sigma) has been studied. The extract from the leaves of Serratula coronata was added to the water of animals of experimental groups 2 and 3 (n = 8 in each group) daily at the dose of 5 and 15 mg phytoecdysteroids per kg of body weight respectively. Animals of the control group 1 (n = 8) received water alone throughout the experiment. Daily volume of drunk fluid was recorded. At the 15th day of the experiment animals were taken out using the decapitation under the light ether anesthesia. The content of corticosterone, prostaglandin E2 and beta-endorphin in rat blood plasma were determined by ELISA test. Plasma level of noradrenaline was determined by HPLC. DNA damage and percentage of apoptotic cells (apoptotic index) were measured in isolated cells of the thymus, heart and brain by single-cell gel electrophoresis (the comet assay). Significantly lower concentration of norepinephrine was detected in plasma of experimental animals from groups 2 and 3 (10.3 +/- 1.1 and 7.2 +/- 0.8 ng/ml, respectively) compared to the same index in the control group (20.4 +/- 3.4 ng/ml). Significant differences of other biochemical parameters for all groups of animals have not been identified. Statistical significant difference in the ratio of corticosterone/norepinephrine compared with control animals was detected for a group of rats consumed the highest dose of phytoecdysteroids. There was no statistically significant difference in DNA fragmentation and apoptosis index in animals consumed phytoecdysteroids in compare with the control group of animals. The absence of the activity of apoptosis in cells of the heart, brain and thymus of rats treated with phytoecdysteroid extract may indicate the safety of its use in the diet of the animals.

Ecdysteroids, molting hormones of insects, can exert several mild, non-hormonal bioactivities in mammals, including humans. In a previous study, we have found a significant effect of certain derivatives on the ABCB1 transporter mediated multi-drug resistance of a transfected murine leukemia cell line. In this paper, we present a structure-activity relationship study focused on the apolar dioxolane derivatives of 20-hydroxyecdysone. Semi-synthesis and bioactivity of a total of 32 ecdysteroids, including 20 new compounds, is presented, supplemented with their complete 1H- and 13C-NMR signal assignment.

Among various pigmentation patterns on caterpillars, sequential spot markings are often observed and used for aposematic colouration. In contrast to adult wings, caterpillar cuticle markings are repeatedly generated at each moult, but little is known about how the patterns are formed and maintained periodically. Here we focus on a silkworm mutant, multi lunar (L), with twin-spot markings on sequential segments. Positional cloning of L and expression analyses reveal that cis-regulatory change in Wnt1 is responsible for the spot patterning. The periodical upregulation of Wnt1 in response to ecdysteroid is detected only in epidermis within spot marking area. We verify by transgenic expression that the ectopic Wnt1 induces the additional pigmentation. Furthermore, the association of Wnt1 expression with spot markings is observed in the wild Bombyx species and swallowtail butterfly Papilio machaon. Taken together, we anticipate that periodic Wnt1 expression may contribute to natural variations of spot patterning on caterpillar cuticle.

With reference to the ethnopharmacological significance of Vitex doniana Sweet (Lamiaceae) leaves in the treatment of stomach and rheumatic pains as well as inflammatory disorders, biological studies on its stem bark extracts have also reported anti-inflammatory and analgesic activities, with no attempt to identify the active components. Chromatographic and spectroscopic procedures identified three new phytoecdysteroids: 21-hydroxyshidasterone (1), 11β-hydroxy-20-deoxyshidasterone (2), and 2,3-acetonide-24-hydroxyecdysone (3) from the stem bark methanol extracts along with known ecdysteroids shidasterone (4), ajugasterone C (5), 24-hydroxyecdysone (6), and 11β,24-hydroxyecdysone (7). The compounds (1-7) showed significant (p ≤ 0.05) inhibitory effect at 100 mg/kg dose on rat paw oedema development due to carrageenan-induced inflammation in Sprague Dawley rats. These results suggest a possible contribution of ecdysteroids to the anti-inflammatory effect of some V. doniana stem bark extracts., (Georg Thieme Verlag KG Stuttgart · New York.)

The rockpool mosquito, Georgecraigius atropalpus, is a facultatively autogenous species that produces its first egg clutch without a blood meal shortly after emergence. Several days after depositing this clutch, females must take a blood meal to produce a second egg clutch. Decapitation of females shortly after emergence or blood ingestion prevents egg maturation. Here, we report that a single injected dose of the neuropeptide ovary ecdysteroidogenic hormone (OEH) fully restored egg maturation in decapitated females in both circumstances. This neuropeptide and two insulin-like peptides (ILPs) are potent gonadotropins in the related yellow fever mosquito, Aedes aegypti. ILP3 was marginally restorative in decapitated G. atropalpus, and ILP4 had no effect. Egg maturation in non- and blood-fed G. atropalpus was dependent on the enzymatic mobilization of amino acids from stored protein or the blood meal for yolk protein (vitellogenin, VG) synthesis and uptake by oocytes. We further show that OEH stimulates serine protease activity in the fat body of newly eclosed females or in the midgut of blood-fed ones, and ecdysteroid hormone production by the ovaries of both females. In contrast, only 20-hydroxyecdysone stimulated VG synthesis in the fat body of non- and blood-fed females. Using RNA interference to knock down expression of the insulin receptor, we found that OEH still fully restored autogenous egg maturation. In summary, our results identify OEH as a primary regulator of egg maturation in both autogenous and blood-fed G. atropalpus females and suggest the shift from blood meal-dependent to blood meal-independent release of OEH is a key factor in the evolution of autogeny in this species.