Phytoecdysteroids from the juice of Serratula coronata L. (Asteraceae).

Seven phytoecdysteroids have been isolated from Serratula coronata L. One of them is a new phytoecdysteroid, 3-epi-20-hydroxyecdysone. Two further ecdysteroids, 20-hydroxyecdysone 22-acetate and taxisterone, are isolated from this species for the first time in addition to the typical S. coronata ecdysteroids, 20-hydroxyecdysone, ecdysone, ajugasterone C and polypodine B. The juice squeezed from aerial parts of fresh plants of S. coronata was extracted with ethyl acetate. The ecdysteroids were isolated by a combination of chromatographic techniques (mainly HPLC) and identified by 1D and 2D (1)H and (13)C NMR experiments and mass-spectrometry. The biological activities of 3-epi-20-hydroxyecdysone (EC(50)=1.6 x 10(-7) M), taxisterone (EC(50)=9.5 x 10(-8) M) and ajugasterone C (EC(50)=6.2 x 10(-8) M) have been determined in the Drosophila melanogaster B(II) bioassay for ecdysteroid agonist activity.     

A comparative analysis of the proteins from a cell culture and from field plants of the ecdysteroid producer Serratula coronata L.]

Differences in the composition and amount of proteins synthesized in the cell culture and leaves of field plants Serratula coronata have been shown. They proceed from differences in intensity of synthesis of secondary metabolites, ecdysteroids, whose content in the cell culture is considerably lower.     

Effect of vitamin D3, arginine, and a biologically active complex from Serratula coronata on free radical oxidation of lipids in vitamin D deficiency

Lipid free-radical oxidation (LFRO) on the D-deficient model with the animal administration per os by vitamin D3 (cholecalciferol), specimen from Serratula coronata plant, containing low molecular weight biological sterol--20-hydroxyecdysone (ecdysterone) was investigated. As well influence of arginine as one of the amino acid components of the specimen from Serratula coronata was investigated. The LFRO in the blood serum, liver microsomal and mitochondrial fractions were characterize by measured of the chemiluminescence kinetic parameters. Vitamin D3, the specimen from Serratula coronata plant and arginine displayed some antioxidant (AO) properties as was established. On the basis of the received experimental data the series of their antioxidizing activity: vitamin D3 = drug from Serratula coronata > arginine were offered. The possible mechanism of the 20-hydroxyecdysone and arginine participation on LFRO maybe in free-radical reactions breaking via hydroxy and amino groups in structure of their molecules.     

Investigation of ecdysteroid content and composition of Silene repens indigenous in Mongolia and introduced into western Siberia

The ecdysteroid profile was investigated by HPLC, and it has been found that 2-deoxyecdysone, 2-deoxy-20-hydroxyecdysone, polypodine B, 20-hydroxyecdysone, and integristerone A are synthesized in the aerial part. The composition of biologically active substances (BAS) has been investigated, and the presence of triterpene glycosides, alkaloids and flavonoids has been confirmed; tannins have been detected for the first time. It has been shown that the ecological conditions, such as altitude above sea level and humidity, influence the ecdysteroid composition and BAS levels.     

Inheritance of resistance in smooth bromegrass to the crown rust fungus

Common smooth bromegrass (Bromus inermis Leyss.) is octoploid, 2n = 8x = 56, with a genome structure of AAAAB1B1B2B2. Tetrasomic inheritance patterns have been observed in smooth bromegrass, but disomic inheritance is also expected from cytologic observations. Smooth bromegrass is susceptible to the crown rust fungus (Puccinia coronata Corda.). The objective of this study was to determine the inheritance of smooth bromegrass resistance to P. coronata. Seven smooth bromegrass clones, three susceptible and four resistant, were selfed and crossed in a diallel with bulked reciprocals. Inoculations were made with a population of P. coronata from PL-BDR1 smooth bromegrass. Resistance of smooth bromegrass to this population of P. coronata is complex. At least three genes appear to be involved in this host-pathogen interaction, one tetrasomic dominant gene which determines susceptibility (S) and two dominant genes (R1 and R2) that may be complementary and could be inherited either tetrasomically or disomically. Other genes may be involved in the smooth bromegrass-P. coronata interaction, possibly accounting for the lack of fit to expected ratios of some progeny. Heterogeneity for avirulence phenotype in the pathogen population may also have contributed to lack of fit of some progeny. Multiple resistance genes were detected because a pathogen population, likely consisting of genotypes with different genes for virulence, was used to challenge the host.     

Self-fertility and other distinguishing characteristics of a new morphotype of Puccinia coronata pathogenic on smooth brome grass

A new morphotype of Puccinia coronata, pathogenic to Bromus inermis, a common roadside and pasture grass in the northern United States, was discovered in the 1990s and described as P. coronata f. sp. bromi by Delgado et al in 2001. Puccinia coronata f. sp. bromi does not require fertilization of pycnia to produce aecia on its alternate host, whereas fertilization is required in all other varieties or formae speciales of P. coronata with aecial hosts in the family Rhamnaceae and for which life cycles have been described. Promycelia of P. coronata f. sp. bromi produce only two basidiospores, and each receives a pair of nuclei from the promycelium. The nuclei divide again so that mature basidiospores each contain four nuclei. Puccinia coronata f. sp. bromi has smaller teliospores than P. coronata var. avenae, and its substomatal vesicles are non-septate and distinctly shaped compared to those of P. coronata var. avenae. In addition, nuclei of P. coronata f. sp. bromi contain less DNA than those of P. coronata var. avenae. Puccinia coronata f. sp. bromi is further distinguished from P. coronata var. avenae and P. coronata var. hordei in being avirulent on both oat and barley, whereas neither P. coronata var. avenae nor P. coronata var. hordei are virulent on Bromus inermis.     

Additional minor ecdysteroid components of Leuzea carthamoides

Seventeen additional minor ecdysteroid compounds were isolated and identified from the roots of Leuzea carthamoides (Wild.) DC. Eight of them are new phytoecdysteroids: carthamoleusterone (13) is a new side-chain cyclo-ether with five-membered ring; 14-epi-ponasterone A 22-glucoside (12) is a rare and unusual natural 14 beta-OH epimer; 15-hydroxyponasterone A (11) is also new and rare with its C-15 substituted position, as well as 22-deoxy-28-hydroxymakisterone C (18) possessing secondary hydroxyl in position C-28 and 26-hydroxymakisterone C (20) with hydroxy groups in positions 25 and 26. New are also 1 beta-hydroxymakisterone C (21) and 20,22-acetonides of inokosterone (8) and integristerone A (10). Series of already known ecdysteroids: ecdysone (1), 20-hydroxyecdysone 2- and 3-acetates (3 and 4), turkesterone (6), inokosterone (7), 24-epi-makisterone A (14), and amarasterone A (22) are reported here as new constituents of L. carthamoides. Seven earlier reported Leuzea ecdysteroids: 20-hydroxyecdysone (2), ajugasterone C (5), integristerone A (9), 24(28)-dehydromakisterone A (15), 24(28)-dehydroamarasterone B (16), (24Z)-29-hydroxy-24(28)-dehydromakisterone C (17) and makisterone C (19) are also included because they are now better characterized.     

华麻花头根中的蜕皮甾酮类成分

从华麻花头(Serratula chinensis S．Moore)根中分得7种蜕皮甾酮类化合物,经光谱分析和化学方法,分别鉴定为：20-羟基蜕皮松(1),podecdysone C(2),3-氧-乙酰基-20-羟基蜕皮松(3),20-羟基蜕皮松-20,22．-缩丁醛(4),shidasterone(5),atrotosterone C(6)和carthamosterone(7),其中20-羟基蜕皮松-20,22．缩丁醛为一新的化合物。     

Phytoecdysteroids from Lamium spp: identification and distribution within plants

Bioassay/radioimmunoassay (RIA) analysis of the seeds of four Lamium species, L. album, L. galeobdolon, L. maculatum and L. pupureum revealed the presence of phytoecdysteroids in all of them. Bioassay/RIA-guided and photo-diode array-monitored HPLC analysis of the aerial parts of L. album and L. purpureum led to the isolation of four known ecdysteroids (abutasterone, inokosterone, polypodine B and pterosterone) from the former, and 20-hydroxyecdysone from the latter. Distribution and identities of ecdysteroids in different parts of these two species and also in the seed extract of L. maculatum have been analysed by RIA and bioassay.     

Antioxidative and free radical scavenging effects of ecdysteroids from Serratula strangulata

The antioxidative and free radical scavenging effects of four ecdysteroids, 20-hydroxyecdysone (E1), 25-deoxy-11,20-dihydroxyecdysone (E2), 24-(2-hydroxyethyl)-20-hydroxyecdysone (E3), and 20-hydroxyecdysone-20,22-monoacetonide (E4), isolated from the Chinese herb Serratula strangulata have been investigated in vitro. These ecdysteroids could protect human erythrocytes against oxidative hemolysis induced by a water-soluble azo initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). They could also inhibit the peroxidation of rat liver microsomes induced by hydroxyl radicals, as monitored by the formation of thiobarbituric acid reactive substances (TBARS), and prevent radical-induced decrease of membrane fluidity as determined by fluorescence polarization. They reacted with galvinoxyl radicals in homogeneous solution, and the pseudo-first-order rate constants were determined spectrophotometrically by following the disappearance of galvinoxyl radicals. Compounds E1 and (or) E3 were the most active in both antioxidative and radical-scavenging reactions.     

Phytoecdysteroids in seeds of Lloydia serotina (Liliaceae)

Seeds of a number of species in the Liliaceae (sensu Brummitt, 1992) were examined for the presence of ecdysteroid agonist and antagonist activities. No species were antagonistic to 20-hydroxyecdysone action on the ecdysteroid-responsive Drosophila melanogaster B_II cell line and only one extract, that of Lloydia serotina, was agonistic. This activity is attributable to the presence of phytoecdysteroids as detected by ecdysteroid-specific radioimmunoassay and the agonist version of the B_II bioassay. HPLC in conjunction with radioimmunoassay and bioassay have been used to determine the ecdysteroid profile. The major ecdysteroids present are identified as 20-hydroxyecdysone and polypodine B (5β,20-dihydroxyecdysone).     

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.     

Ecdysteroid agonist and antagonist activities in species of the Solanaceae

Previously, it has been shown that certain withanolides from Iochroma gesnerioides (Solanaceae) possess ecdysteroid antagonistic activity. Phytoecdysteroids (agonists) are widely distributed in the plant world, but solanaceous species have not been extensively examined for their presence. We have now surveyed 128 species of solanaceous plants for the presence of ecdysteroid agonist and antagonist activities using the Drosophila melanogaster B(II) cell line bioassay. Only weak antagonistic activity was associated with a few of the methanolic extracts, including those from species known to contain high levels of withanolides. Therefore, the major withanolides are inactive per se, but they may be activated after ingestion by invertebrate predators. Several extracts possessed ecdysteroid agonist activity as a consequence of the presence of phytoecdysteroids. Phytoecdysteroid-accumulating species are at least as common in the Solanaceae as they are in plants in general. Preliminary characterization of the identities of the phytoecdysteroids present in the most active extracts has been performed by hplc separations on normal- and reversed-phase systems in conjunction with ecdysteroid-specific radioimmunoassay and bioassay. Each of the phytoecdysteroid-accumulating species examined (Browallia speciosa, Nierembergia hippomanica var violacea, N. solanacea and Solanum nigrum) contain a cocktail of ecdysteroids, of which 20-hydroxyecdysone and polypodine B (5beta,20-dihydroxyecdysone) are major components.     

Growth-promoting effects of ecdysteroids and juvenile hormone on in vitro development of the larval endoparasitoid,Venturia canescens (Hymenoptera: Ichneumonidae)

We previously reported that lipophorin, fetal bovine serum (FBS), and 20-hydroxyecdysone (20-HE) are essential for the development of the larval endoparasitoid Venturia canescens larvae in vitro. The present study was undertaken to determine the optimal concentrations of those three substances in the MGM-450 medium, and to examine the hormonal effects of ecdysteroids and juvenile hormone (JH) on the development of the parasitoid larvae in vitro. When the culture was started with embryos at the post-germband stage, concentrations of 3 mg/ml of lipophorin and 20% of FBS were most suitable for the development of the parasitoid. The growth-promoting effect of 20-HE increased in a concentration-dependent manner and peaked at a concentration of 1 &mgr;g/ml. Excess concentration led to malformations of the larvae. Three other ecdysteroids, ecdysone, 2-deoxy-20-hydroxyecdysone, and polypodine B had the same effect, although their activity was lower than that of 20-HE. Cholesterol had no effect; most larvae failed to develop. When the medium was supplemented with JH, the duration of the developmental period was significantly shortened, but this hormone was not found to be essential.     

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.     

Ecological relations of agricultural populations of ecdysteroid-containing plants Rhaponticum carthamoides (Willd.) Iljin and Serratula coronata L. with herbivorous insects report 2. Composition variability of phytoecdysteroids in agrocenoses and their role in the vulnerability of plants to phytophagans

The accumulation and variability of ecdysteroids, which are analogs of the insect molting hormones, were studied during ontogeny of agricultural populations of Rhaponticum carthamoides (Leuzea carthamoides DC.) and Serratula coronata with relation to the plant age and cultivation conditions. The physiological role of ecdysteroids in the ecological interactions with pests was evaluated. It was found that the enhancement of herbivore activity coincided with biochemical changes in the composition of ecdysteroids having different physiological activities and was accompanied by damage to reproductive organs. During ontogenetic (age-related) changes and seasonal development in the vegetation season, the content of the physiologically active ecdysteroid 20-hydroxyecdysone decreased and relatively moderately active inokosterone and weakly active ecdysone were accumulated in reproductive shoots.     

A possible role of 20-hydroxyecdysone in embryonic development of the silkworm Bombyx mori

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various ecdysteroids and the amounts of these ecdysteroids fluctuate during embryonic development. In order to know the function of egg ecdysteroids in embryonic development of B. mori, we examined the biological activities of various egg ecdysteroids by in vitro ligand-binding assay and bioassay using B. mori eggs. First, using the ecdysteroid receptor of B. mori (BmEcR-B1/BmUSP heterodimer) prepared by yeast and Escherichia coli expression systems, the interaction between the ecdysteroid receptor and various egg ecdysteroids of B. mori was analyzed. The relative binding affinities of egg ecdysteroids to the BmEcR-B1/BmUSP heterodimer decreased in the order of 20-hydroxyecdysone > 2-deoxy-20-hydroxyecdysone > 22-deoxy-20-hydroxyecdysone > ecdysone > 2-deoxyecdysone > ecdysone 22-phosphate. Next, several egg ecdysteroids of B. mori were injected into the prospective diapause eggs, which show a very low level of free ecdysteroids at the onset of embryonic diapause (gastrula stage). Approximately 7% of them (P < 0.002, chi(2)-test) developed beyond the gastrula stage without entering diapause by the injection of 20-hydroxyecdysone (25 ng/egg). In contrast, the injection of other ecdysteroids was not effective in inducing embryonic development. These results suggest that 20-hydroxyecdysone, via the ecdysteroid receptor, is responsible for the developmental difference between diapause and non-diapause in B. mori embryos. Furthermore, it was suggested that continuous supply of 20-hydroxyecdysone may be required to induce embryonic development.     

Synthesis and phosphorylation of ecdysteroids during ovarian development in the silkworm, Bombyx mori

In the silkworm, Bombyx mori, it has been demonstrated that most free ecdysteroids in the ovary are converted to physiologically inactive ecdysteroid 22-phosphates, which are then transformed back to free ecdysteroids during early embryonic development. Two specific enzymes involved in the reciprocal conversion of ecdysteroids, namely, ecdysteroid 22-kinase (EcKinase) and ecdysteroid-phosphate phosphatase, have been isolated and characterized. In this study, we first attempted a phylogenetic analysis of EcKinase. The resulting phylogenetic tree showed that many proteins homologous to B. mori EcKinase are found not only in ecdysozoa, including insects and nematodes, but also in teleosts, fungi, and bacteria. We then investigated the sites where free ecdysteroids are synthesized and phosphorylated in the ovary. We found that (1) the mRNAs of two P450 enzymes involved in ecdysteroidogenesis, CYP306a1 (25-hydroxylase) and CYP314a1 (20-hydroxylase), are expressed mainly in follicle cells, (2) EcKinase mRNA localizes in the oocyte and nurse cells, and (3) EcKinase immunoreactivity localizes mainly in the external region of the oocyte, not in nurse cells or follicle cells. From these results, we suggest that ecdysteroids in the B. mori ovary are synthesized in follicle cells and transferred into the oocyte, where they are phosphorylated by EcKinase, whose mRNA originates from nurse cells and the oocyte itself.     

The identification and titres of conjugated and free ecdysteroids in developing ovaries and newly-laid eggs of Schistocerca gregaria

Ecdysteroid levels throughout ovarian development and in newly-laid eggs of S. gregaria have been determined. A simple method for the separation of free and conjugated ecdysteroids is described. Both free and polar conjugated ecdysteroids are present at the end of oögenesis and in newly-laid eggs, but the polar conjugated ecdysteroids always predominate; 95% of the total ecdysteroid in newly-laid eggs is in the conjugated form. Ecdysone, 2-deoxyecdysone and 20-hydroxyecdysone have been fully characterized from both the ‘free’ and ‘conjugated’ fractions. The presence of traces of 26-hydroxyecdysone in the ‘conjugate’ fraction was indicated by HPLC analyses. The levels of ecdysteroid released from the conjugates of newly-laid eggs were 35 μg/egg pod (44 μg/g wet weight) for ecdysone, 16 μg/egg pod (19.4 μg/g) for 2-deoxyecdysone and 5 μg/egg pod (6.1 μg/g) for 20-hydroxyecdysone. The level of free ecdysone found in newly-laid eggs was 2 μg/egg pod (2.6 μg/g).     

浑善达克沙地无芒雀麦（Bromus inermis）空间分布格局

通过西北-东南和东北-西南方向两条宽5m×长3 km的样带和一块4m×4m的样方调查,研究了浑善达克沙地无芒雀麦、植被、土壤水分及土壤盐分的空间变异特点及其相互关系。结果表明,样带内植被盖度、无芒雀麦盖度、土壤水分和土壤盐分的空间变化格局相似,然而,在不同的空间方向上,它们具有不同的空间变化;样方内无芒雀麦地上部生物量、分株数和土壤盐分具有相似的空间变化格局,但却不同于植被地上部总生物量和土壤水分的空间变化格局。相关分析表明,样带内植被盖度、无芒雀麦盖度与土壤水分和土壤盐分之间均具有显著的正相关关系。土壤水分和土壤盐分也具有显著的正相关。样方内无芒雀麦地上部生物量、分株数及植被地上部总生物量与土壤水分均具有显著的正相关关系,但与土壤盐分没有显著的相关关系。无芒雀麦地上部生物量、分株数和植被地上部总生物量三者之间,以及土壤水分和土壤盐分之间也均具有显著的正相关关系。研究结果表明了植被与土壤之间的相互关系依赖于空间尺度。