Tamara Savchenko

MOSCHAMIN, CIS-MOSCHAMINE, MOSCHAMINDOLE AND MOSCHAMINDOLOL: FOUR NOVEL INDOLE ALKALOIDS FROM CENTAUREA MOSCHATA

Abstract Four novel indole alkaloids: (E) N-(3-methoxy-4-hydroxycinnamoyl)-5-hydroxytryptamine (moschamine, 1), (Z) N-(3-methoxy-4-hydroxycinnamoyl)-5-hydroxytryptamine (cis-moschamine, 2), {2-(3-methoxy-4-hydroxyphenyl)}-dihydrofuro[kl]-1H-pyrrolo[[fg]-2-oxo-1,2,3,4,5,6-hexahydro-3-benzazocine (moschamindole, 3) and 1-(3-methoxy-4,7-dihydroxybenzyl)-10-hydroxy-1H-Pyrrolo[fg]-2-oxo-1,2,3,4,5,6-hexahydro-3-benzazocine (moschamindolol, 4) have been isolated from the seeds of Centaurea moschata. The structures of these compounds have been determined primarily on the basis of extensive 1D and 2D NMR experiments.

Plant natural products as insect steroid receptor agonists and antagonists

Findings to date on plant secondary compounds which possess ecdysteroid-like or anti-ecdysteroid activities in an efficient and effective bioassay based on an ecdysteroid-responsive insect cell-line are summarised. Several novel antagonists have been identified, among which the cucurbitacins are the best characterised and have been shown to compete with ecdysteroids for the ligand binding site of the insect steroid hormone receptor. Certain withanolides, limonoids and resveratrol derivatives also antagonise 20-hydroxyecdysone action. Additionally, several new phytoecdysteroids have been isolated and identified. In common with all other ecdysteroids, these possess agonistic activity in the B II bioassay. Extensive SAR studies based on the potencies of a large number of purified ecdysteroids have been performed and molecular (CoMFA) modelling used to characterise ecdysteroid binding to the ligand binding site of the receptor complex.

Phytoecdysteroids in the genus Asparagus (Asparagaceae)

Phytoecdysteroids, plant steroids which are analogues of invertebrate steroid hormones, probably contribute to the deterrence of phytophagous invertebrate predators. They also seem to possess antimicrobial activity and several pharmaceutical and medicinal benefits have been ascribed to them. Here. we present a survey of seeds of 16 species of the genus Asparagus (Asparagaceae), including the crop species A. officinalis, for ecdysteroid agonists (including phytoecdysteroids) and antagonists. Seven species were found to contain ecdysteroids with levels ranging from just detectable (A. racemosus and A. sarmentosus) to relatively high (A. laricinus). RP-HPLC/RIA/bioassay has been used to separate positive extracts of four species (A. falcatus, A. laricinus, A. ramosissimus and A. scandens) and analyse the ecdysteroid profiles. The identities of the major ecdysteroids were confirmed by NP-HPLC. Seeds of A. officinalis do not contain detectable levels of ecdysteroids, but leaves, stems and roots contain low levels (detectable by RIA). This indicates that A. officinalis retains the genetic capacity to synthesise ecdysteroids and that future strategies could be developed for enhanced protection of asparagus spears through elevated ecdysteroid levels.

Distribution of phytoecdysteroids in the Caryophyllaceae.

Certain genera within the Caryophyllaceae (especially Silene and Lychnis) have received a significant amount of attention with regard to the isolation and identification of ecdysteroids. However, the taxonomy of this family is difficult. Hence, the occurrence of phytoecdysteroids in members of the Caryophyllaceae is presented, and combined with new data on ecdysteroid agonist (phytoecdysteroid) and antagonist activities, in order to survey the distribution of phytoecdysteroid-containing species within this large family, and to assess the utility of phytoecdysteroids as chemotaxonomic markers. The new data presented (representing ca. 110 species) have been obtained by the application of sensitive biological/biochemical methods for the detection of ecdysteroid agonists and antagonists, using Drosophila melanogaster B(II) bioassay and ecdysteroid-specific immunoassays. In the antagonist version of the B(II) bioassay, only weak ecdysteroid antagonist activities were detected in a few of the extracts. From both new and previously available data, it was found that phytoecdysteroids were present predominantly in the Genera Lychnis, Petrocoptis, Sagina and Silene. Comparison of ecdysteroid occurrence with a molecular phylogeny for the tribe Sileneae [Taxon 44 (1995) 525] revealed close association of ecdysteroid occurrence with certain groups of this tribe. In 14 species of Silene examined, there is a reasonable, but not absolute, relationship between the presence of ecdysteroids in the seeds and in other plant parts. Where ecdysteroids are present in the plant, highest concentrations are generally present in the roots.

Two limonoids from Turraea obtusifolia (Meliaceae), prieurianin and rohitukin, antagonise 20-hydroxyecdysone action in a Drosophila cell line

Bioassay-assisted HPLC analyses yielded two prieurianin-type limonoids, prieurianin and rohitukin, from the seeds of Turraea obtusifolia, which act as antagonists of 20-hydroxyecdysone action in the Drosophila melanogaster BII cell line. With a 20-hydroxyecdysone concentration of 5 x 10(-8) M, the ED50 values for prieurianin and rohitukin are 10(-5) M and 1.25 x 10(-4) M, respectively.

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.

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.

DISTRIBUTION AND IDENTITIES OF PHYTOECDYSTEROIDS IN THE GENUS BRIZA (GRAMINEAE)

Within the genus Briza, phytoecdysteroids are predominantly associated with native Eurasian species, while none of the native South American species tested was found to accumulate detectable levels of ecdysteroids in their seeds. Bioassay/RIA-guided HPLC analysis led to the isolation of six known ecdysteroids (abutasterone, ecdysone, 20-hydroxyecdysone, polypodine B, pterosterone and sidisterone) from the seeds of Briza maxima. The structures of these compounds were elucidated by UV, EIMS and a series of 1D and 2D NMR experiments, notably 1H NMR, 13C PENDANT, COSY 45, HMBC, HMQC and NOESY. The distributions of ecdysteroids present in different plant parts of Briza spp. were analysed by RIA. Those species containing detectable levels of ecdysteroids in the seeds (B. elatior, B. maxima, B. media and B. minima) possessed even higher levels of ecdysteroids in the plants, while the other species, with undetectable ecdysteroid levels in the seeds, possessed very low (probably biologically insignificant) levels in the plants. The highest levels of ecdysteroids are associated with meristematic region at the root/aerial interface in plants of B. elatior.

Phytoecdysteroids in the Genus Agapanthus (Alliaceae)

Bioassay/RIA-based analysis of seeds of 15 species, subspecies and varieties of Agapanthus revealed the presence of phytoecdysteroids in most of them. However, the concentrations varied markedly between samples. The accumulation of phytoecdysteroids was highest in a sample of A. campanulatus ssp. patens and a sample of A. inapertus ssp. hollandii (c. 1.5 mg ecdysone equivalents per g seed, using the DBL-1 antiserum). Moderate levels were present in a sample of A. inapertus ssp. inapertus. Phytoecdysteroid levels were almost undetectable in samples of A. coddii, A. caulescens ssp. angustifolius, A. minor var. alba and A. praecox ssp. orientalis. RP-HPLC separation coupled with monitoring by RIA and bioassay revealed that the samples contain significantly different ecdysteroid profiles. The major phytoecdysteroid in several of the extracts has been identified as 20-hydroxyecdysone on the basis of chromatographic evidence. It is concluded that ecdysteroid levels and profiles vary considerably in seeds of the genus Agapanthus, even between seed samples of the same species.

Chemotaxonomic significance of ecdysteroid agonists and antagonists in the Ranunculaceae: phytoecdysteroids in the genera Helleborus and Hepatica

We present here a survey of ca. 100 species within 16 genera of the family Ranunculaceae for the presence of ecdysteroid agonist and antagonist activities in methanolic seed extracts. The levels of phytoecdysteroids (agonists) have been quantified by radioimmunoassay and bioassay. A few samples possess weak antagonistic activity. Phytoecdysteroids are most prominently associated with the genus Helleborus. In this genus, species fall into two distinct classes: those with low or undetectable ecdysteroid levels and those with high ecdysteroid levels. The relationship between ecdysteroid levels and the biology of the plants in this genus is discussed. Additionally, the extract of Hepatica triloba Chaix seeds contains a significant level of phytoecdysteroids. Several other species contain low levels of phytoecdysteroids, as detected by radioimmunoassay. Together with our previous data on the genera Anemone and Pulsatilla, this survey allows us to present an overview of the distribution of ecdysteroids in this family.