A 20-hydroxyecdysone-enriched fraction from Pfaffia glomerata (Spreng.) pedersen roots alleviates stress, anxiety, and depression in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Pfaffia glomerata roots are widely used in Brazil to treat various pathological conditions, particularly psychological disorders. 20-hydroxyecdysone, a phytosteroid present in the plant, can promote greater body resistance against exogenous and endogenous stressors. The objective of this study was to evaluate the possible neuroprotective effect of a 20-hydroxyecdysone-enriched fraction (20E-EF), obtained from P. glomerata roots, in an acute murine stress model. MATERIAL AND METHODS: The 20E-EF was obtained by partitioning the methanol extract from P. glomerata roots with dichloromethane. Mice were treated by gavage with three doses of 20E-EF (3, 10, and 30 mg/kg) and parameters of stress, anxiety, and depression were evaluated. Biomarkers of oxidative stress (enzymes, antioxidant profile, and oxidized molecules) were evaluated in the cortex, striatum (basal ganglia), and hippocampus of animals treated with 30 mg/kg of 20E-EF. RESULTS: Mass spectrometry revealed that 20E was the main compound in the dichloromethane fraction. At a dose of 30 mg/kg, 20E-EF reduced stress, anxiety, and depression, while stimulating antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase), promoting antioxidant activity (antioxidant capacity, sulfhydryl groups, and reduced glutathione), and reducing oxidative markers (lipid peroxidation). In addition, 20E increased the concentration of NO in the striatum, possibly improving memory function and antioxidant activity. CONCLUSION: A 30 mg/kg dose of 20E-EF was able to reduce stress, anxiety, and depression, in addition to maintaining antioxidant defenses of the cortex and striatum. These findings open new perspectives for understanding the therapeutic properties of P. glomerata and the underlying mechanism(s).

Combined Use of Deep Eutectic Solvents, Macroporous Resins, and Preparative Liquid Chromatography for the Isolation and Purification of Flavonoids and 20-Hydroxyecdysone from Chenopodium quinoa Willd.

: Deep eutectic solvents (DESs) were used in combination with macroporous resins to isolate and purify flavonoids and 20-hydroxyecdysone from Chenopodium quinoa Willd by preparative high-performance liquid chromatography (HPLC). The extraction performances of six DESs and the adsorption/desorption performances of five resins (AB-8, D101, HPD 400, HPD 600, and NKA-9) were investigated using the total flavonoid and 20-hydroxyecdysone extraction yields as the evaluation criteria, and the best-performing DES (choline chloride/urea, DES-6) and macroporous resin (D101) were further employed for phytochemical extraction and DES removal, respectively. The purified extract was subjected to preparative HPLC, and the five collected fractions were purified in a successive round of preparative HPLC to isolate three flavonoids and 20-hydroxyecdysone, which were identified by spectroscopic techniques. The use of a DES in this study significantly facilitated the preparative-scale isolation and purification of polar phytochemicals from complex plant systems.

Two new phytoecdysteroids from Sphenocentrum jollyanum Pierre root.

The crude methanol extract of Sphenocentrum jollyanum root exhibited 98% and 80% antimicrobial activity against Aspergillus fumigatus Pinh and Vancomycin resistant enterococcus (VRE) at a concentration of 200 µg/mL, with IC50 11.45 and 12.95 µg/mL, respectively. The ethyl acetate fraction of methanol extract showed in-vitro antimicrobial activity against A. fumigatus Pinh at 83% with IC50 of <8 µg/mL. The phytochemical investigation of ethyl acetate fraction yielded six compounds, which were identified by their NMR, IR and MS spectral analyses as two new phytoecdysteroidal glycosides Sphenocentroside A (1), and Sphenocentroside B (2), and four known phytoecdysteroids: polypodoaurein (3), polypodine B (4), ecdysterone (5), and 20, 26-dihydroxyecdysone (6).

Obtaining functional powder tea from Brazilian ginseng roots: Effects of freeze and spray drying processes on chemical and nutritional quality, morphological and redispersion properties.

In this work, the aqueous extract obtained from Brazilian ginseng (Pfaffia glomerata) roots (BGR), rich in beta-ecdysone and fructooligosaccharides (FOS), was powdered by spray drying and freeze drying techniques aiming to obtain a novel functional food product. The effects of these drying techniques on the chemical and nutritional quality, morphological and redispersion properties of the BGR powders were evaluated. The BGR powders obtained by both spray drying and freeze drying techniques maintained their beta-ecdysone and FOS contents after drying, demonstrating the stability of these functional compounds. It was found that the wettability of the powders obtained by different treatments was affected by the drying technique because freeze-dried particles reached the lower values (66 ±â€¯5 s) while spray-dried particles showed a greater time for dispersion into water (150 ±â€¯25 s). This behavior was mainly associated with differences between powder morphological properties since the freeze-dried particles presented a more porous structure, resulting in a greater water diffusivity into microstructure during the redispersion process. Drying process did not affect the storage stability of powders because the glass transition temperature (Tg) for both samples was approximately 160 °C at a relative humidity of 56%. Thus, both BGR powders presented adequate redispersion properties to constitute a new functional tea or even to be used as a functional ingredient in food products.

Bioavailability and structural study of 20-hydroxyecdysone complexes with cyclodextrins.

20-Hydroxyecdysterone - (2ß,3ß,5ß,22R)-2,3,14,20,22,25-hexahydroxycholest-7-en-6-one was isolated in satisfactory yield using ethanol extraction from the aerial part of Silene wolgensis (Hornem.) Otth; sometimes Silene wolgensis (Willd.) Bess. ex Spreng. The complexation of the phytoecdysteroid with ß-cyclodextrin was studied by NMR spectroscopy. By studying the changes in chemical shifts of protons of substrates and receptors it was found that ecdysterone interacts with cyclodextrins to form supramolecular inclusion complexes of stoichiometric composition of 1:1 or 1:2. Ecdysterone-ß-cyclodextrin complexes exhibit 100 times higher solubility in water than the parent compound.

Ponasterone A and F, Ecdysteroids from the Arctic Bryozoan Alcyonidium gelatinosum.

A new ecdysteroid, ponasterone F (1) and the previously reported compound ponasterone A (2) were isolated from specimens of the Arctic marine bryozoan Alcyonidium gelatinosum collected at Hopenbanken, off the coast of Edgeøya, Svalbard. The structure of 1 was elucidated, and the structure of 2 confirmed by spectroscopic methods including 1D and 2D NMR and analysis of HR-MS data. The compounds were evaluated for their ability to affect bacterial survival and cell viability, as well as their agonistic activities towards the estrogen receptors α and ß. The compounds were not active in these assays. Compound 2 is an arthropod hormone controlling molting and are known to act as an allelochemical when produced by plants. Even though its structure has been previously reported, this is the first time a ponasterone has been isolated from a bryozoan. A. gelatinosum produced 1 and 2 in concentrations surpassing those expected of hormonal molecules, indicating their function as defence molecules against molting predators. This work adds to the chemical diversity reported from marine bryozoans and expanded our knowledge of the chemical modifications of the ponasterones.

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.

Ecdysterones from Rhaponticum carthamoides (Willd.) Iljin reduce hippocampal excitotoxic cell loss and upregulate mTOR signaling in rats.

Glutamate-induced excitotoxicity is a key pathological mechanism in many neurological disease states. Ecdysterones derived from Rhaponticum carthamoides (Willd.) Iljin (RCI) have been shown to alleviate glutamate-induced neuronal damage; although their mechanism of action is unclear, some data suggest that they enhance signaling in the mechanistic target of rapamycin (mTOR) signaling pathway. This study sought to elucidate the mechanisms underlying ecdysterone-mediated neuroprotection. We used in silico target prediction and simulation methods to identify putative ecdysterone binding targets, and to specifically identify those that represent nodes where several neurodegenerative diseases converge. We then used histological analyses in a rat hippocampal excitotoxicity model to test the effectiveness of ecdysterones in vivo. We found that RCI-derived ecdysterones should bind to glutamatergic NMDA-type receptors (NMDARs); specifically, in vivo modeling showed binding to the GRIN2B subunit of NMDARs, which was found also to be a node of convergence in several neurodegenerative disease pathways. Computerized network construction by using pathway information from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed putative links between GRIN2B and mTOR pathway elements including phosphoinositide-3kinase (PI3K), mTOR, and protein kinase C (PKC); these elements are associated with neuronal survival. Brain tissue western blots of ecdysterone-treated rats showed upregulated PI3K, Akt, mTOR, and phosphorylated Akt and mTOR, and down regulated GRIN2B and the apoptotic enzyme cleaved caspase-3. Ecdysterone treatment also prevented glutamate-induced rat hippocampal cell loss. In summary, RCI-derived ecdysterones appear to prevent glutamatergic excitotoxicity by increasing mTOR/Akt/PI3K signaling activity.

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.

Nitric Oxide and ERK mediates regulation of cellular processes by Ecdysterone.

The complex process of wound healing is a major problem associated with diabetes, venous or arterial disease, old age and infection. A wide range of pharmacological effects including anabolic, anti-diabetic and hepato-protective activities have been attributed to Ecdysterone. In earlier studies, Ecdysterone has been shown to modulate eNOS and iNOS expression in diabetic animals and activate osteogenic differentiation through the Extracellular-signal-Regulated Kinase (ERK) pathway in periodontal ligament stem cells. However, in the wound healing process, Ecdysterone has only been shown to enhance granulation tissue formation in rabbits. There have been no studies to date, which elucidate the molecular mechanism underlying the complex cellular process involved in wound healing. The present study, demonstrates a novel interaction between the phytosteroid Ecdysterone and Nitric Oxide Synthase (NOS), in an Epidermal Growth Factor Receptor (EGFR)-dependent manner, thereby promoting cell proliferation, cell spreading and cell migration. These observations were further supported by the 4-amino-5-methylamino- 2' ,7' -difluorofluorescein diacetate (DAF FM) fluorescence assay which indicated that Ecdysterone activates NOS resulting in increased Nitric Oxide (NO) production. Additionally, studies with inhibitors of both the EGFR and ERK, demonstrated that Ecdysterone activates NOS through modulation of EGFR and ERK. These results clearly demonstrate, for the first time, that Ecdysterone enhances Nitric Oxide production and modulates complex cellular processes by activating ERK1/2 through the EGF pathway.

Oxidized Metabolites of 20-Hydroxyecdysone and Their Activity on Skeletal Muscle Cells: Preparation of a Pair of Desmotropes with Opposite Bioactivities.

Increasing the activation of protein kinase B (Akt) has been suggested as a key signaling step in the nonhormonal anabolic activity of the phytoecdysteroid 20-hydroxyecdysone (20E) in mammals. Base-catalyzed autoxidation of this compound was shown previously to yield interesting B-ring-modified analogues. Herein is reported a thorough study on this reaction, resulting in the preparation and complete NMR spectroscopic assignments of calonysterone (5) and its previously overlooked desmotropic pair (7), along with two new sensitive metabolites of 20E. The two isomers showed considerable stability in solution. Time dependency of the reaction for yield optimization is also presented; by means of analytical HPLC, the two desmotropes can reach a maximum combined yield of >90%. The activity of these compounds on Akt phosphorylation was tested in murine skeletal muscle cells. Compounds 2 and 5 showed more potent activity than 20E in increasing Akt activation, while compound 7 exerted an opposite effect. As such, the present study provides the first direct evidence for a pair of desmotropes exerting significantly different bioactivities.

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.

[BRAIN FOCAL ISCHEMIA-REPERFUSION CAUSES A DECREASED RESISTANCE OF ERYTHROCYTES FROM VENOUS BLOOD TO ACID HEMOLYSIS, WHICH IS PREVENTED BY ECDYSTERONE].

We investigated the resistance of erythrocytes from rat brain venous blood to acid hemolysis in the dynamics of brain ischemic period (15, 30, 45 and 60 min), as well as in the early (5 min) and distant (24h) period of brain reperfusion. Brain ischemia-reperfusion was made in rats that received ecdysterone (standartized extract of Serratula coronata) within 18 days (per os, 1 mg/kg). Analysis of the kinetic curves of acid hemolysis showed a pronounced (60 times, from 1.45 to 85.85% at 60 min of brain ischemia and at 5 min of brain reperfusion, respectively) increase of unstable erythrocytes that hemolyzed easily (< 2.5 min). In the preconditioned rats, this increase was only 8-fold. During the period of brain ischemia, with a maximum at 15th minute, in the venous blood from brain the diene conjugates (DK) pools increased from 2.40 to 9.48 ng/mg protein and LTC4 pools increased from 1.49 to 5.98 pmol/mg protein. Even more pools of DC and LTC4 were increased at 5th min of brain reperfusion. In animals received ecdysterone, during ischemia and early reperfusion period, both pools of DC and LTC4 in venous blood were lower than that in the controls. The latter implies a possible antiradical mechanism of the protective effect of ecdysterone.

Quinoa seeds leach phytoecdysteroids and other compounds with anti-diabetic properties.

Quinoa (Chenopodium quinoa Willd.) contains high levels of biologically active phytoecdysteroids, which have been implicated in plant defense from insects, and have shown a range of beneficial pharmacological effects in mammals. We demonstrated that the most prevalent phytoecdysteroid, 20-hydroxyecdysone (20HE), was secreted (leached) from intact quinoa seeds into water during the initial stages of seed germination. Leaching efficiency was optimized by ethanol concentration (70% ethanol), temperature (80°C), time (4h), and solvent ratio (5 ml/g seed). When compared to extraction of macerated seeds, the leaching procedure released essentially all the 20HE available in the seeds (491 µg/g seed). The optimized quinoa leachate (QL), containing 0.86% 20HE, 1.00% total phytoecdysteroids, 2.59% flavonoid glycosides, 11.9% oil, and 20.4% protein, significantly lowered fasting blood glucose in obese, hyperglycemic mice. Leaching effectively releases and concentrates bioactive phytochemicals from quinoa seeds, providing an efficient means to produce a food-grade mixture that may be useful for anti-diabetic applications.

Tetra-acetylajugasterone a new constituent of Vitex cienkowskii with vasorelaxant activity.

Tetra-acetylajugasterone C (TAAC) was found to be one of the naturally occurring compounds of the Cameroonian medicinal plant Vitex cienkowskii which is responsible for a vasorelaxant activity of an extract of this plant. The evaluation of the underlying mechanisms for the relaxing effect of TAAC was determined using aortic rings of rats and mice. TAAC produced a concentration-dependent relaxation in rat artery rings pre-contracted with 1µM noradrenaline (IC50: 8.40µM) or 60mM KCl (IC50: 36.30µM). The nitric oxide synthase inhibitor l-NAME (100µM) and the soluble guanylate cyclase inhibitor ODQ (10µM) significantly attenuated the vasodilatory effect of TAAC. TAAC also exerted a relaxing effect in aorta of wild-type mice (cGKI(+/+); IC50=13.04µM) but a weaker effect in aorta of mice lacking cGMP-dependent protein kinase I (cGKI(-/-); IC50=36.12µM). The involvement of calcium channels was studied in rings pre-incubated in calcium-free buffer and primed with 1µM noradrenaline prior to addition of calcium to elicit contraction. TAAC (100µM) completely inhibited the resulting calcium-induced vasoconstriction. The same concentration of TAAC showed a stronger effect on the tonic than on the phasic component of noradrenaline-induced contraction. This study shows that TAAC, a newly detected constituent of Vitex cienkowskii contributes to the relaxing effect of an extract of the plant. The effect is partially mediated by the involvement of the NO/cGMP pathway of the smooth muscle but additionally inhibition of calcium influx into the cell may play a role.

[20-Hydroxyecdysone--plant adaptogen: an anabolic effect, possible use in sports nutrition].

In the review the presentation about plant adaptogens--biologically active compounds is given. Its administration can help to achieve non-specific state of high resistance. The hypothetical mechanism of action: adaptogens are prostressors, reducing excessive increase of stress mediators in the following stress exposure. The features of adaptogenic effect of phytoecdysteroids, polyhydroxylated sterols, which are analogs of hormones of molting and metamorphosis of arthropodas, and are structurally similar to glucocorticoids on the example of the most widely studied phytoecdysteroid--20-hydroxyecdysone--are described. The results of studies of anabolic action of 20-hydroxyecdysone in experiments on laboratory animals and the possible explanation (existing in the modern scientific literature) of the mechanism of this phenomenon are discussed. Scientific publication testifying on the application of phytoecdysteroids to remove chronic fatigue syndrome, reducing nerve and muscle fatigue, improve memory and attention processes are presented. The prospects of using the 20-hydroxyecdysone in the composition of food supplements and specialized products for athletes are discussed.

LC/MS/MS identification of 20-hydroxyecdysone in a scorpion (Liocheles australasiae) and its binding affinity to in vitro-translated molting hormone receptors.

Recent advances in mass spectrometry (MS) technology have facilitated the detection and quantification of minor components in organisms and the environment. In this study, we successfully identified 20-hydroxyecdysone (20E) in first instar nymphs (7 days after hatching) of the scorpion Liocheles australasiae, using tandem mass spectrometry combined with high-performance liquid chromatography (LC/MS/MS). This substance was not found in adults after the fifth stage. Other possible molting hormone candidates such as makisterone A (MaA) and ponasterone A (PoA), both of which are reported to be the molting hormones of a few arthropod species, were not detected in this scorpion. The ligand-receptor binding of 20E and its analogs was quantitatively evaluated against the in vitro-translated molting hormone receptor, the heterodimer of ecdysone receptor (EcR) and the retinoid X receptor (RXR) of L. australasiae (LaEcR/LaRXR). The concentrations of ecdysone (E), MaA, 20E, and PoA that are required to inhibit 50% of [(3)H]PoA binding to the LaEcR/LaRXR complex were determined to be 1.9, 0.69, 0.05, and 0.017 µM, respectively. The activity profiles of these 4 ecdysteroids are consistent with those obtained for the molting hormone receptors of several insects. The binding of a non-steroidal E agonist, tebufenozide, to EcR was not observed even at high concentrations, indicating that the structure of the ligand-binding pocket of LaEcR is not favorable for interaction with tebufenozide.

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.

Phytoecdysteroids from the rhizomes of Brainea insignis.

Phytoecdysteroid glucosides, brainesterosides A-E, were isolated from the rhizomes of Brainea insignis along with three known phytoecdysteroids, ponasteroside A, ponasterone A, and 20-hydroxyecdysone. Their structures were elucidated by spectroscopic and chemical means. A possible biogenetic pathway is postulated for these compounds. The chemosystematic significance of ponasterone A is discussed.

[Studies on the chemical constituents from the roots of Tinospora sagittata var. yunnanensis (II)].

OBJECTIVE: To study the chemical constituents from the roots of Tinospora sagittata var. yunnanensis. METHODS: Various chromatographic techniques were used to isolate and purify the constituents. The structures of these compounds were elucidated on the basis of spectral analysis. RESULTS: Seven compounds were isolated from the plant and their structures were identified as tinophylloloside (1), epitinophylloloside (2), 2-deoxycrustecdysone (3), polypodine B (4) , (+)-5'-methoxyisolariciresinol 3alpha-O-beta-D-glucopyranoside (5), geniposide (6), adenine (7). CONCLUSION: All compounds are isolated from the plant for the first time,and compounds 4, 5, 6 and 7 are isolated firstly from the genus.