Analytical strategy for the detection of ecdysterone and its metabolites in vivo in uPA(+/+)-SCID mice with humanized liver, human urine samples, and estimation of prevalence of its use in anti-doping samples.

Ecdysteroids are of interest as potential sport performance enhancers, due to their anabolic effects. The current study aimed to analyze levels of the most abundant ecdysteroid, ecdysterone (20-hydroxyecdysone, 20-OHE) in easily available dietary supplements, and, outline an analytical strategy for its detection, and that, of its metabolites, (1) following administration of pure 20-OHE to uPA(+/+)-SCID mice with humanized liver, (2) in a human volunteer after ingestion of two supplements, one with a relatively low, and the other a high, concentration of 20-OHE, and, (3) to estimate the prevalence of use of 20-OHE in elite athletes (n = 1000). Of the 16 supplements tested, only five showed detectable levels of 20-OHE, with concentrations ranging from undetectable up to 2.3 mg per capsule. Urine of uPA(+/+)-SCID urine showed the presence of 20-OHE and its metabolite, 14 deoxy ecdysterone, within 24 hours (hr) of ingestion. In humans, both the parent and the metabolite were detectable within 2 to 5 hr of ingestion, with the metabolite being detectable for longer than the parent. After ingestion of a low dose supplement, the parent and metabolite were detectable for 70 and 48 hr, while following the higher dose it was 96 and 48 hr, respectively. Analysis of urines from athletes (n = 1000) confirmed four positives for 20-OHE, suggesting a prevalence of use of 0.4%. Prevalence of its use by elite athletes was relatively low, however, this needs to be confirmed in other populations, and with other related ecdysteroids.

BmNPV-induced hormone metabolic disorder in silkworm leads to enhanced locomotory behavior.

Many parasites alter the host locomotory behaviors in a way that increases their fitness and progeny transmission. Baculoviruses can manipulate host physiology and alter the locomotory behavior by inducing 'hyperactivity' (increased locomotion) or 'tree-top disease' (climbing high up to the top before dying). However, the detailed molecular mechanism underlying virus-induced this hyperactive behavior remains elusive. In the present study, we showed that BmNPV invaded into silkworm brain tissue, resulting in severe brain damage. Moreover, BmNPV infection disturbed the insect hormone balance. The content of 20-hydroxyecdysone (20E) in hemolymph was much lower during the hyperactive stage, while the dopamine (DA) titer was higher than mock infection. Exogenous hormone treatment assays demonstrated that 20E inhibits virus-induced ELA (enhanced locomotory activity), while dopamine stimulates this behavior. More specificity, injection of dopamine or its agonist promote this hyperactive behavior in BmNPV-infected larvae. Taking together, our findings revealed the important role of hormone metabolism in BmNPV-induced ELA.

Blue and red light affects morphogenesis and 20-hydroxyecdisone content of in vitro Pfaffia glomerata accessions.

The combination of different colors from light-emitting diodes (LEDs) may influence growth and production of secondary metabolites in plants. In the present study, the effect of light quality on morphophysiology and content of 20-hydroxyecdysone (20E), a phytoecdysteroid, was evaluated in accessions of an endangered medicinal species, Pfaffia glomerata, grown in vitro. Two accessions (Ac22 and Ac43) were cultured in vitro under three different ratios of red (R) and blue (B) LEDs: (i) 1R:1B, (ii) 1R:3B, and (iii) 3R:1B. An equal ratio of red and blue light (1R:1B) increased biomass accumulation, anthocyanin content, and 20E production (by 30-40%). Moreover, 1R:1B treatment increased the size of vascular bundles and vessel elements, as well as strengthened xylem lignification and thickening of the cell wall of shoots. The 1R:3B treatment induced the highest photosynthetic and electron transport rates and enhanced the activity of oxidative stress-related enzymes. Total Chl content, Chl/Car ratio, and NPQ varied more by accession type than by light source. Spectral quality affected primary metabolism differently in each accession. Specifically, in Ac22 plants, fructose content was higher under 1R:1B and 1R:3B treatments, whereas starch accumulation was higher under 1R:3B, and sucrose under 3R:1B. In Ac43 plants, sugars were not influenced by light spectral quality, but starch content was higher under 3R:1B conditions. In conclusion, red and blue LEDs enhance biomass and 20E production in P. glomerata grown in vitro.

How reliable is dietary supplement labelling?-Experiences from the analysis of ecdysterone supplements.

The present study aimed to design, develop, and optimize an analytical procedure to perform the quantitative determination of ecdysterone in commercially available dietary supplements. The newly developed procedure is based on the extraction of ecdysterone from the supplements and the subsequent analysis by an optimized UHPLC-MS/MS method. Chromatographic separation was performed on an Agilent Eclipse Plus C18 column (2.1 mm x 100 mm, particle size 1.8 µm). The mass spectrometer was operated in positive ionization mode (ESI+) with acquisition in dynamic multiple reaction monitoring (dMRM) mode. Using the protonated molecular ion [M+H]+ ecdysterone (target) and cortisol (internal reference) were detected at m/z 481 and 363, respectively. The assay was fully validated according to ICH guidelines and the method resulted to be fit for purpose in terms of accuracy and precision (CV% and RE% <15). Time-different intermediate precision was found within the reported range according to AOAC guideline for dietary supplements and botanicals. Quantitation has been performed using an external calibration considering the minimal matrix influences, preliminarily assessed following a cross comparison with an elaborate and time consuming standard addition method. The method was successfully applied to 12 different dietary supplements labelled to contain ecdysterone, showing an actual content generally much lower than the labelled one.

Steroid-Enriched Fraction of Achyranthes bidentata Protects Amyloid ß Peptide 1-40-Induced Cognitive Dysfunction and Neuroinflammation in Rats.

The roots of Achyranthes bidentata Blume (AB) is commonly used in the treatment of osteoporosis and dementia in traditional Chinese medicine. Pharmacological reports evidenced that AB possessed anti-osteoarthritis effects. However, there is little literature about the anti-dementia activities of AB. The present study was designed to prepare steroid-enriched fraction of AB (ABS) and investigate whether ABS can protect from cognitive dysfunction and neuroinflammation against Aß 1-40-induced Alzheimer's disease (AD) model in rats. ABS only contained 135.11 ± 4.28 mg of ecdysterone per gram. ABS (50 mg/kg) reversed the dysfunction of exploratory activity and memory function on plus-maze and Morris water maze caused by Aß 1-40 in rats. ABS (50 mg/kg) also decreased amyloid deposition, neurofibrillary tangle, neural damage, activated astrocyte, and microglial caused by Aß 1-40. Furthermore, ABS reversed the phenomenon of neural oxidative damage and neuroinflammation, including the higher levels of MDA and cytokines, and the lower activities of antioxidant enzymes and GSH levels caused by Aß 1-40 in rat cortex and hippocampus. Finally, ABS restored the activation of ERK pathway and decreased NF-κB phosphorylation and translocation altered by Aß 1-40. ABS alone (50 mg/kg) promoted cognitive function, activated brain antioxidant defense system, and decreased brain TNF-α levels in sham group. Therefore, ABS has the cognition-promoting and antidementia potential. Steroids especial ecdysterone are major active components of AB. The action mechanism is due to decreasing oxidative stress and neuroinflammation through modulating ERK pathway, NF-κB phosphorylation, and translocation in Aß 1-40-induced AD rat model.

Porous organic cage incorporated monoliths for solid-phase extraction coupled with liquid chromatography-mass spectrometry for identification of ecdysteroids from Chenopodium quinoa Willd.

Here, a porous organic cage (POC)-incorporated polymeric monolith was fabricated in a syringe through the introduction of the POC into poly(ethylene glycol dimethacrylate) monolith in a one-step traditional free-radical polymerization proceess. The resulting monolithic phases were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), powder X-ray diffraction (PXRD), nitrogen adsorption/desorption experiments and thermogravimetric analysis (TGA), which confirmed the successful incorporation of the POC in the monolithic matrix. The functionality of the POC-incorporated poly(EDMA) monolith facilitated for the solid phase extraction (SPE) of 20-hydroxecdysone (an ecdysteroid) from Chenopodium quinoa Willd. extract coupled with UPLC-QqQ-MS/MS, exhibiting satisfactory accuracy (93-106%), precision (< 6.5%) and reusability. In addition, UPLC-Q-Exactive-Orbitrap-MS/MS analysis of the quinoa sample after SPE by POC-incorporated monolith provided the identification of 20-hydroxecdysone and three other ecdysteroids. These results demonstrate the potential of POC-incorporated monoliths for the SPE of ecdysteroids from complex plant systems.

Extraction optimization and UHPLC method development for determination of the 20-hydroxyecdysone in Sida tuberculata leaves.

Sida tuberculata (ST) is a Malvaceae species widely distributed in Southern Brazil. In traditional medicine, ST has been employed as hypoglycemic, hypocholesterolemic, anti-inflammatory and antimicrobial. Additionally, this species is chemically characterized by flavonoids, alkaloids and phytoecdysteroids mainly. The present work aimed to optimize the extractive technique and to validate an UHPLC method for the determination of 20-hydroxyecdsone (20HE) in the ST leaves. Box-Behnken Design (BBD) was used in method optimization. The extractive methods tested were: static and dynamic maceration, ultrasound, ultra-turrax and reflux. In the Box-Behnken three parameters were evaluated in three levels (-1, 0, +1), particle size, time and plant:solvent ratio. In validation method, the parameters of selectivity, specificity, linearity, limits of detection and quantification (LOD, LOQ), precision, accuracy and robustness were evaluated. The results indicate static maceration as better technique to obtain 20HE peak area in ST extract. The optimal extraction from surface response methodology was achieved with the parameters granulometry of 710 nm, 9 days of maceration and plant:solvent ratio 1:54 (w/v). The UHPLC-PDA analytical developed method showed full viability of performance, proving to be selective, linear, precise, accurate and robust for 20HE detection in ST leaves. The average content of 20HE was 0.56% per dry extract. Thus, the optimization of extractive method in ST leaves increased the concentration of 20HE in crude extract, and a reliable method was successfully developed according to validation requirements and in agreement with current legislation.

Ecdysteroid-containing food supplements from Cyanotis arachnoidea on the European market: evidence for spinach product counterfeiting.

Phytoecdysteroids like 20-hydroxyecdysone ("ecdysterone") can exert a mild, non-hormonal anabolic/adaptogenic activity in mammals, and as such, are frequently used in food supplements. Spinach is well-known for its relatively low ecdysteroid content. Cyanotis arachnoidea, a plant native in China, is among the richest sources of phytoecdysteroids, and extracts of this plant are marketed in tons per year amounts via the internet at highly competitive prices. Here we report the investigation of a series of food supplements produced in Germany and claimed to contain spinach extracts. Twelve ecdysteroids including two new compounds were isolated and utilized as marker compounds. A comparative analysis of the products with Cyanotis and spinach extracts provides evidence that they were manufactured from Cyanotis extracts instead of spinach as stated. Based on the chromatographic fingerprints, 20-hydroxyecdysone 2- and 3-acetate are suggested as diagnostic markers for related quality control. This case appears to represent an unusual type of dietary supplement counterfeiting: undeclared extracts from alternative plants would supposedly 'guarantee' product efficacy.

UVB Radiation Delays Tribolium castaneum Metamorphosis by Influencing Ecdysteroid Metabolism.

Ultraviolet B (UVB) radiation is an important environmental factor. It is generally known that UVB exhibits high genotoxicity due to causing DNA damage, potentially leading to skin carcinogenesis and aging in mammals. However, little is known about the effects of UVB on the development and metamorphosis of insects, which are the most abundant terrestrial animals. In the present study, we performed dose-response analyses of the effects UVB irradiation on Tribolium castaneum metamorphosis, assessed the function of the T. castaneum prothoracicotropic hormone gene (Trcptth), and analyzed ecdysteroid pathway gene expression profile and ecdysterone titers post-UVB irradiation. The results showed that UVB not only caused death of T. castaneum larvae, but also delayed larval-pupal metamorphosis and reduced the size and emergence rate of pupae. In addition, we verified the function of Trcptth, which is responsible for regulating metamorphosis. It was also found that the expression profiles of Trcptth as well as ecdysteroidogenesis and response genes were influenced by UVB radiation. Therefore, a disturbance pulse of ecdysteroid may be involved in delaying development under exposure to irradiation. To our knowledge, this is the first report indicating that UVB can influence the metamorphosis of insects. This study will contribute to a better understanding of the impact of UVB on signaling mechanisms in insect metamorphosis.

Analysis of phytochemical variations in dioecious Tinospora cordifolia stems using HPLC/QTOF MS/MS and UPLC/QqQLIT -MS/MS.

INTRODUCTION: The stem of dioecious Tinospora cordifolia (Menispermaceae) is a commonly used traditional Ayurvedic medicine in India having several therapeutic properties. OBJECTIVE: To develop and validate LC-MS methods for the identification and simultaneous quantitation of various secondary metabolites and to study metabolomic variations in the stem of male and female plants. METHODS: Ethanolic extract of stems were analysed by HPLC/ESI-QTOF-MS/MS for rapid screening of bioactive phytochemicals. High resolution MS and MS/MS in positive ESI mode were used for structural investigation of secondary metabolites. An UPLC/ESI-QqQ(LIT) -MS/MS method in MRM mode was developed and validated for the simultaneous quantitation of five bioactive alkaloids. RESULTS: Identification and characterisation of 36 metabolites including alkaloids, sesquiterpenes and phytoecdysteroids were performed using LC-MS and MS/MS techniques. The bioactive alkaloids such as jatrorrhizine, magnoflorine, isocorydine, palmatine and tetrahydropalmatine were successfully quantified in male and female plants. The mean abundances of magnoflorine jatrorrhizine, and oblongine were significantly (P < 0.05) higher in male plants while mean abundances of tetrahydropalmatine, norcoclaurine, and reticuline were significantly (P < 0.05) higher in female plants. CONCLUSIONS: Phytochemicals in the stem of male and female Tinospora cordifolia showed significant qualitative and quantitative variations. LC-MS and MS/MS methods can be used to differentiate between male and female plants based on their chemical profiles and quantities of the marker bioactive alkaloids. This chemical composition difference was also evident during vegetative stage when there were no male and female flowers.

The minor ecdysteroids from Ajuga turkestanica.

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

Quantitative determination of juvenile hormone III and 20-hydroxyecdysone in queen larvae and drone pupae of Apis mellifera by ultrasonic-assisted extraction and liquid chromatography with electrospray ionization tandem mass spectrometry.

In this paper, a method for the rapid and sensitive analysis of juvenile hormone III (JH III) and 20-hydroxyecdysone (20E) in queen larvae and drone pupae samples was presented. Ultrasound-assisted extraction provided a significant shortening of the leaching time for the extraction of JH III and 20E and satisfactory sensitivity as compared to the conventional shake extraction procedure. After extraction, determination was carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS) operating in electrospray ionization positive ion mode via multiple reaction monitoring (MRM) without any clean-up step prior to analysis. A linear gradient consisting of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid, and a ZORBAX SB-Aq column (100 mm × 2.1 mm, 3.5 µm) were employed to obtain the best resolution of the target analytes. The method was validated for linearity, limit of quantification, recovery, matrix effects, precision and stability. Drone pupae samples were found to contain 20E at concentrations of 18.0 ± 0.1 ng/g (mean ± SD) and JH III was detected at concentrations of 0.20 ± 0.06 ng/g (mean ± SD) in queen larvae samples. This validated method provided some practical information for the actual content of JH III and 20E in queen larvae and drone pupae samples.

Supercritical fluid extraction of cynaropicrin and 20-hydroxyecdysone from Leuzea carthamoides DC.

Leuzea carthamoides is an adaptogenic plant containing biologically active compounds as ecdysteroids and guaianolide-type sesquiterpene lactones, conventionally extracted from the plant with ethanol. It may be a potential source of the mentioned natural compounds. Ethanol-modified near-critical CO(2) was used as selective solvent with the aim to increase the level of 20-hydroxyecdysone in the extract from L. carthamoides roots and to remove selectively cynaropicrin, a sesquiterpene lactone of bitter taste, from the leaves. The extraction conditions were varied (pressure 20-28 MPa, temperature 40-60 degrees C, ethanol concentration in the solvent 0-7.1%) and the extraction yield and extract composition were compared with the results of ethanolic extraction. The supercritical fluid extraction (SFE) from finely powdered plant was controlled by phase equilibrium. Cynaropicrin was quantitatively removed from the leaves where 89% of 20-hydroxyecdysone was retained. The extraction yield of 20-hydroxyecdysone from roots with ethanol-modified CO(2 )was lower by 30% than with ethanol but its concentration in the extract was higher by 67%.

Supercritical fluid extraction of ecdysterone from the roots of Achyranthes bidentata BL.

Ecdysterone has been found in a great many plants and animals and has some valuable pharmaceutical properties. The present study was conducted to investigate optimal conditions for the extraction of the compound by supercritical fluid extraction from the roots of Achyranthes bidentata BL. An orthogonal array design (OAD), OA(9)(3(4)), was employed as a chemometric method for optimization of the extraction of ecdysterone from the herbal medicine. Four parameters, namely, pressure and temperature of the supercritical fluid, the dynamic extraction time, and the flow rate of dimethyl sulfoxide, were studied and optimized by a three-level OAD. Determinations of the extracts were performed by high-performance liquid chromatography. The effects of the parameters were studied using analysis of variance. The results shown that the yield of ecdysterone could be influenced by the four parameters to a similar degree. The yield for DMSO-modified supercritical CO(2) was in the range from 0.65 to 1.03 mg/g under the selected conditions. In comparison with methanol-modified supercritical CO(2 )and Soxhlet extraction, a higher yield was obtained when DMSO-modified supercritical CO(2) was used.

Preparative-scale chromatography of ecdysteroids: a class of biologically active steroids.

A simple separation procedure is developed for the isolation of the main phytoecdysteroid 20-hydroxyecdysone from the herb Silene viridiflora. The purification in four steps uses only a simple preparative-scale separation technique (i.e., liquid-liquid extraction, precipitation, solid-phase extraction on octadecyl silica, and crystallization). This procedure is extended using classical normal-phase liquid column chromatography, rotation planar chromatography, and preparative high-performance liquid chromatography for the isolation of the minor ecdysteroids: integristerone A, 26-hydroxypolypodine B, 2-deoxy-20,26-dihydroxyecdysone, and polypodine B. 2-Deoxy-20,26-dihydroxyecdysone is isolated from this species for the first time. The isolation of these ecdysteroids in adequate amounts makes them readily available for insect physiology experiments and for structure-activity relationship studies. The preparative-scale separation work also results in a minor, as yet unknown ecdysteroid.

Transcription factor broad suppresses precocious development of adult structures during larval-pupal metamorphosis in the red flour beetle, Tribolium castaneum.

Broad (br), a transcription factor containing the Broad-Tramtrack-Bric-a-brac (BTB) and zinc finger domains was shown to mediate 20-hydroxyecdysone (20E) action and pupal development in Drosophila melanogaster and Manduca sexta. We determined the key roles of br during larval-pupal metamorphosis using RNA interference (RNAi) in a coleopteran insect, Tribolium castaneum. Two major peaks of T. castaneum broad (Tcbr) mRNA, one peak at the end of feeding stage prior to the larvae entering the quiescent stage and another peak during the quiescent stage were detected in the whole body and midgut tissue dissected from staged insects. Expression of br during the final instar larval stage is essential for successful larval-pupal metamorphosis, because, RNAi-mediated knock-down of Tcbr during this stage derailed larval-pupal metamorphosis and produced insects that showed larval, pupal and adult structures. Tcbr dsRNA injected into the final instar larvae caused reduction in the mRNA levels of genes known to be involved in 20E action (EcRA, E74 and E75B). Tcbr dsRNA injected into the final instar larvae also caused an increase in the mRNA levels of JH-response genes (JHE and Kr-h1b). Knock-down of Tcbr expression also affected 20E-mediated remodeling of midgut during larval-pupal metamorphosis. These data suggest that the expression of Tcbr during the final instar larval stage promotes pupal program while suppressing the larval and adult programs ensuring a transitory pupal stage in holometabolous insects.

Sex-specific mediation of foraging in the shore crab, Carcinus maenas.

Experiments were conducted to investigate the sex-specific differences to feeding responses of the shore crab Carcinus maenas throughout the year. Results demonstrate that female shore crabs exhibit stronger feeding responses than males throughout the year with a significantly reduced feeding response in males during the summer months' reproductive season. We also studied the possible function(s) of the moulting hormone, 20-hydroxyecdysone (Crustecdysone) that has been described as a potential female-produced sex pheromone to initiate male reproductive behaviour in a number of crustaceans. We recently presented evidence that for shore crabs this is not the case and now show that the steroid is instead functioning as a sex-specific feeding deterrent protecting the moulting 'soft' female crabs. Whilst male shore crabs were deterred from prey (Mytilus edulis) and synthetic feeding stimulants glycine and taurine when these feeding stimulants were spiked with crustecdysone, intermoult female crabs were significantly less affected and rarely deterred from feeding. This sex specificity of the moulting hormone, in combination with the female sex pheromone, which has no anti-feeding properties, ensures that male crabs mate with soft-shelled, moulted females rather than engage in cannibalism, such as found frequently in cases when soft-shelled females are exposed to intermoult females.

Effects of octopamine on reproduction, juvenile hormone metabolism, dopamine, and 20-hydroxyecdysone contents in Drosophila.

The effect of an experimentally increased octopamine content (feeding flies with OA) on the levels of juvenile hormone (JH) degradation, dopamine (DA), and 20-hydroxyecdysone (20E) contents, oogenesis, and fecundity of wild type Drosophila flies has been studied. OA feeding of the flies was found to (1) cause a considerable decrease in JH degradation in females, but not males, of D. melanogaster and D. virilis; (2) have no effect on DA content in D. melanogaster and D. virilis; (3) increase 20E contents in D. virilis females; (4) decrease to a large extent the number of vitellogenic (stages 8-10) and mature (stage 14) oocytes in D. virilis; and (5) decrease the fecundity of D. melanogaster and D. virilis. A possible mechanism of action of OA as a neurohormone on the reproductive function of Drosophila is discussed.

Dopamine and octopamine regulate 20-hydroxyecdysone level in vivo in Drosophila.

The effects of increased level of dopamine (DA) (feeding flies with DA precursor, L-dihydroxyphenylalanine, L-DOPA) on the level of 20-hydroxyecdysone (20E) and on juvenile hormone (JH) metabolism in young (2-day-old) wild type females (the strain wt) of Drosophila virilis have been studied. Feeding the flies with L-DOPA increased DA content by a factor of 2.5, and led to a considerable increase in 20E level and a decrease of JH degradation (an increase in JH level). We have also measured the levels of 20E in the young (1-day-old) octopamineless females of the strain Tbetah(nM18) and in wild type females, Canton S, of D. melanogaster. The absence of OA led to a considerable decrease in 20E level (earlier it was shown that in the Tbetah(nM18) females, JH degradation was sharply increased). We have studied the effects of JH application on 20E level in 2-day-old wt females of D. virilis and demonstrated that an increase in JH titre results in a steep increase of 20E level. The supposition that biogenic amines act as intermediary between JH and 20E in the control of Drosophila reproduction is discussed.

New phytoecdysteroids from cultured plants of Ajuga nipponensis Makino.

An extract from aerial parts of Ajuga nipponensis Makino was examined by high performance liquid chromatography for minor ecdysteroids. Along with the compounds already reported, namely cyasterone, ajugasterone C, cyasterone 22-acetate and 22-dehydrocyasterone, the presence of three additional bands with the expected ecdysteroid-like UV absorption was observed. The structures of the isolates were unambiguously elucidated based on extensive NMR spectral studies (one and two-dimensional experiments) and pointed out three new phytoecdysteroids. One of the new compounds, 22-dehydrocyasterone 2-glucoside is just the second example of a C-2 glucosyl derivative. The other two compounds displayed hemiacetal functions in the side chain, one unprecedented, and were named ajugacetalsterone A and B.