Harald Greger

Testing of antifungal natural products: methodologies, comparability of results and assay choice

Antifungal testing of filamentous fungi generally suffers from incomparability of results. In order to illustrate this problem, the polyacetylene falcarindiol and the naphthoquinone juglone, two known antifungal natural products, were assayed in various dilution and diffusion bioassays against three selected microfungi, Botrytis cinerea, Cladosporium herbarum and Fusarium avenaceum. Broth microdilution, based on the 96-well microtitre plate format, can be scored by various direct and biochemical methods. As examples, direct observation with image analysis and the fluorescein diacetate scoring method are compared. Of the other methodologies used, results obtained by thin-layer bioautography and the radial growth rate method clearly deviated. Disk diffusion results, however, matched microdilution. In conclusion, microdilution offers the greatest potential of all bioassays to become the future general standard methodology. Copyright

Antitumor Activity and Mechanism of Action of the Cyclopenta[b]benzofuran, Silvestrol

Background Flavaglines are a family of natural products from the genus Aglaia that exhibit anti-cancer activity in vitro and in vivo and inhibit translation initiation. They have been shown to modulate the activity of eIF4A, the DEAD-box RNA helicase subunit of the eukaryotic initiation factor (eIF) 4F complex, a complex that stimulates ribosome recruitment during translation initiation. One flavagline, silvestrol, is capable of modulating chemosensitivity in a mechanism-based mouse model. Methodology/Principal Findings Among a number of flavagline family members tested herein, we find that silvestrol is the more potent translation inhibitor among these. We find that silvestrol impairs the ribosome recruitment step of translation initiation by affecting the composition of the eukaryotic initiation factor (eIF) 4F complex. We show that silvestrol exhibits significant anticancer activity in human breast and prostate cancer xenograft models, and that this is associated with increased apoptosis, decreased proliferation, and inhibition of angiogenesis. We demonstrate that targeting translation by silvestrol results in preferential inhibition of weakly initiating mRNAs. Conclusions/Significance Our results indicate that silvestrol is a potent anti-cancer compound in vivo that exerts its activity by affecting survival pathways as well as angiogenesis. We propose that silvestrol mediates its effects by preferentially inhibiting translation of malignancy-related mRNAs. Silvestrol appears to be well tolerated in animals.

New unsymmetrically substituted tetrahydrofurofuran lignans from artemisia absinthium: Assignment of the relative stereochemistry by lanthanide induced chemical shifts

Abstract The isolation of thirteen tetrahydrofurofuran lignans from the roots of A. absinthium and six closely related species is reported. A new group of four lignans, all stereoisomers of 1- [(3,4-methylenedioxy-5-methoxy) phenyl]-4-(3,4,5-trimethoxyphenyl)-tetrahydro-1H,3H-furo[3,4-c]furan, was found. The compounds, named sesartemin, episesartemin A, episesartemin B, and diasesartemin, were characterized by 1 H NMR (including lanthanide induced shifts), UV, IR and MS. A fifth new lignan of the sesamin type (the eq/eq isomer of fargesin) could be identified as a minor constituent. The relative configurations of two further products (fargesin and epiashantin) could be confirmed by the lanthanide induced shift technique.

Cyclotide Discovery in Gentianales Revisited—Identification and Characterization of Cyclic Cystine-Knot Peptides and Their Phylogenetic Distribution in Rubiaceae Plants

Cyclotides are a unique class of ribosomally synthesized cysteine-rich miniproteins characterized by a head-to-tail cyclized backbone and three conserved disulfide-bonds in a knotted arrangement. Originally they were discovered in the coffee-family plant Oldenlandia affinis (Rubiaceae) and have since been identified in several species of the violet, cucurbit, pea, potato, and grass families. However, the identification of novel cyclotide-containing plant species still is a major challenge due to the lack of a rapid and accurate analytical workflow in particular for large sampling numbers. As a consequence, their phylogeny in the plant kingdom remains unclear. To gain further insight into the distribution and evolution of plant cyclotides, we analyzed ∼300 species of >40 different families, with special emphasis on plants from the order Gentianales. For this purpose, we have developed a refined screening methodology combining chemical analysis of plant extracts and bioinformatic analysis of transcript databases. Using mass spectrometry and transcriptome-mining, we identified nine novel cyclotide-containing species and their related cyclotide precursor genes in the tribe Palicoureeae. The characterization of novel peptide sequences underlines the high variability and plasticity of the cyclotide framework, and a comparison of novel precursor proteins from Carapichea ipecacuanha illustrated their typical cyclotide gene architectures. Phylogenetic analysis of their distribution within the Psychotria alliance revealed cyclotides to be restricted to Palicourea, Margaritopsis, Notopleura, Carapichea, Chassalia, and Geophila. In line with previous reports, our findings confirm cyclotides to be one of the largest peptide families within the plant kingdom and suggest that their total number may exceed tens of thousands.

Tryptamine derived amides from Clausena indica

Abstract Leaf extracts from different Sri Lankan provenances of Clausena indica were compared by HPLC linked with diode array detection. Remarkable chemical differences towards different types of amides were observed between the collections from the northern dry semi-evergreen forests and the central montane rainforests. Apart from the already known phenethyl cinnamide, four new tryptamine derived amides were detected with different acid moieties: the cinnamic acid amides named balasubramide and prebalamide and the isovaleric acid amides named madugin and methylmadugin. Balasubramide is characterised by an eightmembered lactam ring. All structures were established by spectral analyses.

Sulphur-containing amides from Glycosmis species with different antifungal activity

Leaf extracts from different Sri Lankan provenances of Glycosmis mauritiana and G. pentaphylla have been compared by HPLC analyses and tested for antifungal activity. All collections of G. pentaphylla are uniformly characterized by the known alkaloids arborine, skimmianine and arborinine, whereas the different provenances of G. mauritiana differ by displaying chemical polymorphism of different sulphur-containing amides. Three new amides, named illukumbin B, methylillukumbin B and methylillukumbin A, have been isolated and their structures are described on the basis of spectroscopic evidence. They show different antifungal activities in bioautographic tests and appear to be accumulated mainly in individuals collected from more humid and elevated habitats.

Analysis, isolation and insecticidal activity of linear furanocoumarins and other coumarin derivatives fromPeucedanum (Apiaceae: Apioideae).

Peucedanum arenarium Waldst. & Kit.,P. austriacum (Jacq.) Koch,P. coriaceum Reichenb.,P. longifolium Waldst. & Kit,P. officinale L.,P. oreoselinum (L.) Moench,P. ostruthium L., andP. palustre (L.) Moench accumulate different structural types of coumarins including simple coumarins, linear furanocoumarins, linear dihydropyranocoumarins, angular dihydrofuranocoumarins and angular dihydropyranocoumarins. Linear furanocoumarins, known for various biological activities, include some well-known antifeedants, such as bergapten, isopimpinellin, and xanthotoxin. The aim of this investigation was to screen the diverse coumarins fromPeucedanum for insecticidal activity. LC was used to analyze and isolate coumarins for the bioassays. A growth inhibition bioassay with 17 derivatives, comprising all structural types fromPeucedanum, carried out withSpodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) as test organism, indicated the majority of the linear furanocoumarins and the angular dihydrofuranocoumarin athamantin as active compounds. Oxygenation of the prenyl residue of linear furanocoumarins decreased activity. Further formation of an ester with angelic acid even resulted in complete inactivity. Five active linear furanocoumarins, bergapten, isopimpinellin, xanthotoxin, isoimperatorin, and imperatorin, and two linear furanocoumarins with a substituted furan ring, peucedanin and 8-methoxypeucedanin, were compared in a dietary utilization bioassay. Relative growth rate (RGR) and relative consumption rate (RCR) divided the tested coumarins in three groups of similar activity. Isopimpinellin and peucedanin slightly decreased RGR and RCR of the treated larvae, and xanthotoxin, isoimperatorin, and 8-methoxypeucedanin heavily decreased RGR and RCR. Bergapten and imperatorin differed by the lowest RGR values and rather high RCR values. The effects caused by these two coumarins indicate specific postingestive toxicity. The results obtained in this study add to the reputation of coumarins to be an effective chemical defense, postulating that chemical diversity is a necessary trait for well-defended plants.

Aglaia (Meliaceae): An evaluation of taxonomic concepts based on DNA data and secondary metabolites

We performed maximum parsimony and Bayesian analyses (nuclear ITS rDNA, plastid rps16 intron) to estimate phylogenetic relationships within Aglaia (over 100 species in Southeast Asia, the Pacific, and Australia) and its relations among Aglaieae (Meliaceae). Based on 67 accessions of Aglaieae, three taxa of Guareae, and two taxa of Melieae (outgroup), this study provides the first assessment of the current circumscription of Aglaieae, Aglaia, and its sections and to a more limited extent of species concepts in Aglaia. DNA data are compared to recently collected data on chemical profiles. Our analyses indicate (1) the monophyly of Aglaieae; (2) the polyphyly of Aphanamixis; (3) the paraphyly of Aglaia; (4) the existence of at least three entities with respect to Aglaia: (a) the core group of Aglaia section Amoora (dehiscent fruits) with close relationships to Lansium and Reinwardtiodendron, (b) a group comprising morphological intermediates between the two sections, and (c) the core group of Aglaia section Aglaia (indehiscent fruits). Macro- and micromolecular data indicate that complex species are more heterogeneous, i.e., probably containing more than one taxon each, than taxonomically isolated species. A third section in Aglaia is recognized to accommodate A. lawii, A. teysmanniana, and A. beccarii.

Thapsakins: possible biogenetic intermediates towards insecticidal cyclopenta[b]benzofurans from Aglaia edulis

Abstract Nine new flavaglines, cyclopenta[bc]benzopyrans (thapsakins) and benzo[b]oxepines (thapoxepines), were isolated from the lipophilic root extract of Aglaia edulis together with two known cyclopenta[b]benzofurans, aglaroxin A and pannellin. The structures were established on the basis of extensive use of NMR spectroscopic methods (C,H-COSY, NOESY, HMBC, lanthanide induced shifts). Aglaroxin A and pannellin exhibited the strongest insect toxicity toward neonate larvae of Spodoptera littoralis. Possible biogenetic connections between the three skeletal types are outlined and chemosystematic implications of flavagline formation are discussed.

Pyranoquinolones and acridones from Vepris bilocularis

Besides already known furoquinolones and acridones, three new pyrano-2-quinolones and one new acridone were isolated from the leaves of south Indian Vepris bilocularis. The novel alkaloids were identified by spectroscopic means and named 7-methoxyflindersine, 7-prenyloxyflindersine, N-methyl-7-prenyloxy-flindersine and vebilocine.