Phytoecdysteroids from Dianthus superbus L.: Structures and anti-neuroinflammatory evaluation.

Six undescribed C27-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 IC50 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.

Highly Oxidized Ecdysteroids from a Commercial Cyanotis arachnoidea Root Extract as Potent Blood-Brain Barrier Protective Agents.

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 1H and 13C 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.

Ecdysteroids from the Aerial Parts of Paris verticillata.

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.

Two Ecdysteroids Isolated from Micropropagated Lychnis flos-cuculi and the Biological Activity of Plant Material.

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 IC50 of 0.06 mg/mL at the 3rd day of treatment. Both ecdysteroids show comparable activity at IC50 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.

Bioassay-guided isolation and identification of anti-ulcer ecdysteroids from the seeds of Sphenocentrum jollyanum Pierre (Menispermaceae).

Sphenocentrum jollyanum seeds (MeOH extract and n butanol fraction) exhibited urease inhibitory activity (IC50 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 (IC50 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 (IC50 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.

Phytoecdisteroids from Serratula coronata when growing ducklings.

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%.

Phytoecdysteroids from Ajuga iva act as potential antidiabetic agent against alloxan-induced diabetic male albino rats.

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 ß-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.

Isolation and Purification of Three Ecdysteroids from the Stems of Diploclisia glaucescens by High-Speed Countercurrent Chromatography and Their Anti-Inflammatory Activities In Vitro.

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 ß-glucuronidase release in rat polymorphonuclear leukocytes (PMNs) induced by platelet-activating factor. Compounds I-III showed significant anti-inflammatory activities with IC50-values ranging from 1.51 to 11.68 µM, respectively.

Phytoecdysteroids-containing extract from Stachys hissarica plant and its wound-healing activity.

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.

Effects of Phytoecdysteroids (PEDS) Extracted from Cyanotis arachnoidea on Rumen Fermentation, Enzyme Activity and Microbial Efficiency in a Continuous-Culture System.

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.

UV-protective effects of phytoecdysteroids from Microsorum grossum extracts on human dermal fibroblasts.

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.

Assessing herbal products with health claims.

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.

[Phytoecdysteroids influence on the hormonal status and apoptosis in growing rats].

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.

Phytoecdysteroids from the stem bark of Vitex doniana and their anti-inflammatory effects.

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), 11ß-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 11ß,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.

The Karlson Lecture. Phytoecdysteroids: what use are they?

Phytoecdysteroids are analogues of arthropod steroid hormones found in plants, where they deter predation by non-adapted predators. There is potential to exploit this to develop new strategies for pest control, either by using ecdysteroids as lead molecules for the design of novel pest control agents or by alteration of ecdysteroid levels/profiles in crop plants through plant breeding or genetic modification. However, it is other properties of phytoecdysteroids that have led to a rapid recent increase in scientific and commercial interest in these molecules. They are apparently non-toxic to mammals and a wide range of beneficial pharmacological (adaptogenic, anabolic, anti-diabetic, hepatoprotective, immunoprotective, wound-healing, and perhaps even anti-tumour) activities is claimed for them. In particular, this has led to a large (and unregulated) market for ecdysteroid-containing preparations for body-builders, sportsmen, and pets, among others. Ecdysteroids are also being considered as nutraceutical additives to food products. Further, ecdysteroids are good candidates as elicitors for gene-switch systems to be used in medical gene therapy and research applications. In this article, I review the applications of phytoecdysteroids and assess their future potential.

Chemistry and pharmacology of Rhaponticum carthamoides: a review.

Rhaponticum carthamoides (Willd.) Iljin is a perennial herb, commonly known as a maral root or Russian leuzea, which has been used for centuries in eastern parts of Russia for its marked medicinal properties. This review based on 117 literary sources, with many of them being originally published in non-English languages (mainly in Russian), discusses the current knowledge of traditional uses, chemistry, biological effects and toxicity of this species. Several different classes of compounds were previously isolated from various parts of R. carthamoides of which the main groups are steroids, particularly ecdysteroids, and phenolics (flavonoids and phenolic acids) accompanied with polyacetylenes, sesquiterpene lactones, triterpenoid glycosides and terpenes (essential oil). A comprehensive account of the chemical constituents is given in this review (figures of 120 structures are shown). Various types of preparations, extracts and individual compounds derived from this species have been found to possess a broad spectrum of pharmacological effects on several organs such as the brain, blood, cardiovascular and nervous systems as well as on different biochemical processes and physiological functions including proteosynthesis, work capacity, reproduction, and sexual function. Moreover, the extracts and preparations from the plant, which are hopefully safe, exhibited various additional biological effects e.g. antioxidant, immunomodulatory, anticancerogenic, antimicrobial, antiparasitic and insect antifeedant or repellent activities. The results of data analysis on the chemical, pharmacological and toxicological characteristics of R. carthamoides support the view that this species has beneficial therapeutic properties and indicate its potential as an effective adaptogenic herbal remedy. Finally, some suggestions for further research on chemical and pharmacological properties are given in this review.

Selective modulation of P-glycoprotein activity by steroidal saponines from Paris polyphylla.

Bio-guided fractionation of the roots of Paris polyphylla (Trilliaceae), based on inhibition of P-glycoprotein-mediated daunorubicin efflux in K562/R7 cell line, led to isolation and identification of the three saponins 3-O-Rha(1-->2)[Ara(1-->4)]Glc-pennogenine, gracillin and polyphyllin D, and the two ecdysteroids 20-hydroxyecdysone and pinnatasterone. These compounds were tested for multidrug reversion on P-glycoprotein (ABCB1) with both drug-selected and transfected cell lines, and also on Breast Cancer Resistance Protein (BCRP/ABCG2). By contrast to a weak efficiency on BCRP, the three saponins displayed significant effects as inhibitors of P-glycoprotein-mediated drug efflux.

Phytoecdysteroids increase protein synthesis in skeletal muscle cells.

Phytoecdysteroids, which are structurally similar or identical to insect molting hormones, produce a range of effects in mammals, including increasing growth and physical performance. To study the mechanism of action of phytoecdysteroids in mammalian tissue, an in vitro cellular assay of protein synthesis was developed. In C2C12 murine myotubes and human primary myotubes, phytoecdysteroids increased protein synthesis by up to 20%. In vivo, ecdysteroids increased rat grip strength. Ecdysteroid-containing plant extracts produced similar results. The effect was inhibited by a phosphoinositide kinase-3 inhibitor, which suggests a PI3K-mediated mechanism.

Lack of interference of common phytoecdysteroids with production of nitric oxide by immune-activated mammalian macrophages.

Effects of selected common phytoecdysteroids on immunobiological responses triggered by lipopolysaccharide and interferon-gamma (IFN-gamma) were tested under in vitro conditions using murine resident peritoneal macrophages. Namely, production of nitric oxide was investigated. The series of test agents encompassed ecdysteroids occurring often as major components of the ecdysteroid fraction in numerous plant extracts: 20-hydroxyecdysone (20E), polypodine B, ajugasterone C, ponasterone A and inokosterone. Their structural variability concerns only variation in the number and position of hydroxyls. Two additional side-chain modified ecdysteroids: makisterone A (with a methyl substituent at position 24) and carthamosterone (with a cyclic side-chain lactone), and three ecdysteroid analogs: poststerone, rubrosterone and dihydrorubrosterone (devoid of side chains) were included into the test series. All test compounds, except of ponasterone A, represent natural substances isolated from the medicinal plant Leuzea carthamoides and are supposed to be significant for the often reported pharmacological activities of preparations derived from this species. However, the tested ecdysteroids did not interfere with the immunobiological activity of the immunocompetent cells. Our results thus differ from the so far reported information.

Ecdysteroids from Serratula wolffii roots.

Two new natural ecdysteroids, 20,22-didehydrotaxisterone (1) and 1-hydroxy-20,22-didehydrotaxisterone (2), were isolated from the roots of Serratula wolffii. Their structures were elucidated by 1D and 2D NMR spectroscopy and mass spectrometry. The biological activities of these compounds were determined via oral aphid (Acyrthosiphon pisum (Harris)) tests. Compound 1 was inactive and compound 2 exhibited very low toxicity in the oral aphid test. The activities of these two ecdysteroids were in agreement with those of other 22-deoxyecdysteroids.