Dan Staerk

Butenolides from Plant-Derived Smoke: Natural Plant-Growth Regulators with Antagonistic Actions on Seed Germination

Smoke plays an intriguing role in promoting the germination of seeds of many species following a fire. Recently, a bicyclic compound containing a condensed butenolide moiety, 3-methyl-2H-furo[2,3-c]pyran-2-one (1), was reported as a potent germination promoter from plant-derived smoke. In this study, a related butenolide, 3,4,5-trimethylfuran-2(5H)-one (2), which inhibits germination and significantly reduces the effect of 1 when applied simultaneously, was also isolated from plant-derived smoke. The interaction of these compounds with opposing actions on seed germination may have important ecological implications in a post-fire environment and could be useful molecules for understanding the events involved in breaking seed dormancy and promoting seed germination.

In Vitro Plasmodium falciparum Drug Sensitivity Assay: Inhibition of Parasite Growth by Incorporation of Stomatocytogenic Amphiphiles into the Erythrocyte Membrane

ABSTRACT Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC50) of 27.7 ± 0.5 μM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC50 value and the membrane curvature changes caused by lupeol was observed. Preincubation of erythrocytes with lupeol, followed by extensive washing, made the cells unsuitable for parasite growth, suggesting that the compound incorporates into erythrocyte membrane irreversibly. On the other hand, lupeol-treated parasite culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause stomatocyte formation, but not those causing echinocyte formation, were shown to inhibit growth of the parasites, apparently via a mechanism similar to that of lupeol. Since antiplasmodial agents that inhibit parasite growth through erythrocyte membrane modifications must be regarded as unsuitable as leads for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro Plasmodium toxicity assay.

Accelerated dereplication of crude extracts using HPLC–PDA–MS–SPE–NMR: Quinolinone alkaloids of Haplophyllum acutifolium

Direct hyphenation of analytical-scale high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) has been used for accelerated dereplication of crude extract of Haplophyllum acutifolium (syn. Haplophyllum perforatum). This technique allowed fast on-line identification of six quinolinone alkaloids, named haplacutine A-F, as well as of acutine, haplamine, eudesmine, and 2-nonylquinolin-4(1H)-one. Acutine and haplacutine E, isolated by preparative-scale HPLC, showed moderate antiplasmodial activity with IC(50) values of 2.17+/-0.22 microM and 3.79+/-0.24 microM, respectively (chloroquine-sensitive Plasmodium falciparum 3D7 strain).

The Antiparasitic Compound Licochalcone A Is a Potent Echinocytogenic Agent That Modifies the Erythrocyte Membrane in the Concentration Range Where Antiplasmodial Activity Is Observed

ABSTRACT The well-known antiparasitic compound licochalcone A is a potent membrane-active agent that transforms normal erythrocytes into echinocytes in parallel with the inhibition of growth of Plasmodium falciparum cultures, the in vitro antiplasmodial effect apparently being an indirect effect on the host cell. In vitro experiments with synchronous cultures demonstrate that inhibition of invasion is the principal mechanism of growth inhibition. The erythrocyte membrane-modifying effect was also transiently observed in vivo in mice after intravenous administration.

Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis

Abstract Plant‐derived diterpenoids serve as important pharmaceuticals, food additives, and fragrances, yet their low natural abundance and high structural complexity limits their broader industrial utilization. By mimicking the modularity of diterpene biosynthesis in plants, we constructed 51 functional combinations of class I and II diterpene synthases, 41 of which are “new‐to‐nature”. Stereoselective biosynthesis of over 50 diterpene skeletons was demonstrated, including natural variants and novel enantiomeric or diastereomeric counterparts. Scalable biotechnological production for four industrially relevant targets was accomplished in engineered strains of Saccharomyces cerevisiae.

Production of novel fusarielins by ectopic activation of the polyketide synthase 9 cluster in Fusarium graminearum.

Like many other filamentous fungi, Fusarium graminearum has the genetic potential to produce a vast array of unknown secondary metabolites. A promising approach to determine the nature of these is to activate silent secondary metabolite gene clusters through constitutive expression of cluster specific transcription factors. We have developed a system in which an expression cassette containing the transcription factor from the targeted PKS cluster disrupts the production of the red mycelium pigment aurofusarin. This aids with identification of mutants as they appear as white colonies and metabolite analyses where aurofusarin and its intermediates are normally among the most abundant compounds. The system was used for constitutive expression of the local transcription factor from the PKS9 cluster (renamed FSL) leading to production of three novel fusarielins, F, G and H. This group of compounds has not previously been reported from F. graminearum or linked to a biosynthetic gene in any fungal species. The toxicity of the three novel fusarielins was examined against colorectal cancer cell lines where fusarielin H was more potent than fusarielin F and G.

Combining HPLC-PDA-MS-SPE-NMR with circular dichroism for complete natural product characterization in crude extracts: levorotatory gossypol in Thespesia danis.

Despite recent demonstration of the power of HPLC-PDA-MS-SPE-NMR (high-performance liquid chromatography-photodiode-array detection-mass spectrometry-solid-phase extraction-nuclear magnetic resonance) in structure determination of natural products directly from minute amounts of crude extracts, this technique leaves chirality of the compounds uncharacterized. In this work we demonstrate that postcolumn SPE is a useful method of analyte concentration and accumulation not only for NMR but also for CD (circular dichroism) spectroscopy. Thus, use of HPLC-PDA-MS-SPE-NMR in combination with CD allowed rapid detection of ( R)-(-)-gossypol [( R)- 1] in Thespesia danis, providing a very rare example of the predominance of the levorotatory enantiomer of gossypol. Enantioselectivity of the in vitro antiplasmodial activity of gossypol was also demonstrated; the IC50 value of ( R)- 1 was 4.5 +/- 0.2 microM, with the eudismic ratio of about 2.5. No gossypol was detected in Gossypioides kirkii.

Antimalarial sesquiterpene lactones from Distephanus angulifolius.

Combined use of bioassay-guided fractionation based on in vitro antiplasmodial assay and dereplication based on HPLC-PDA-MS-SPE-NMR led to isolation of (6S,7R,8S)-14-acetoxy-8-[2-hydroxymethylacrylat]-15-helianga-1(10),4,11(13)-trien-15-al-6,12-olid and (5R,6R,7R,8S,10S)-14-acetoxy-8-[2-hydroxymethylacrylat]-elema-1,3,11(13)-trien-15-al-6,12-olid, along with vernodalol, vernodalin, and 11,13beta-dihydroxyvernodalin from extract of Distephanus angulifolius. All compounds were identified by spectroscopic methods, including 1D and 2D homo- and heteronuclear NMR experiments. The isolated compounds showed IC(50) values in the range 1.6-3.8 microM and 2.1-4.9 microM against chloroquine sensitive D10 and chloroquine resistant W2 Plasmodium falciparum strains, respectively.

Combining PARAFAC Analysis of HPLC-PDA Profiles and Structural Characterization Using HPLC-PDA-SPE-NMR-MS Experiments: Commercial Preparations of St. John's Wort

Herbal preparations represent very complex mixtures, potentially containing multiple pharmacologically active entities. Methods for global characterization of the composition of such mixtures are therefore of pertinent interest. In this work, chemometric analysis of high-performance liquid chromatography with photodiode-array detection (HPLC-PDA) data from extracts of commercial preparations of Hypericum perforatum (St. Johns wort) that originate from several continents is described. The spectral HPLC profiles were aligned in the elution mode using correlation optimized warping in order to remove peak misalignment caused by retention time shifts due to matrix effects. Furthermore, the warping was assisted by HPLC-PDA-SPE-NMR-MS (SPE = solid-phase extraction) experiments that yielded 1H NMR and 13C NMR data (from 1H-detected heteronuclear correlations), as well as ESI-MS and HRMS data, which enabled the identification of all major mixture constituents. The preprocessed HPLC-PDA data were subjected to parallel factor analysis (PARAFAC), a chemometric method that is a generalization of principal component analysis (PCA) to multi-way data arrays. PCA of the peak areas obtained from the PARAFAC analysis was used to facilitate sample comparison and allowed straightforward interpretation of constituents responsible for the differences in composition between individual preparations. In addition, loadings from the PARAFAC analysis provided pure elution profiles and pure UV spectra even for coeluting peaks, thus enabling the identification of chromatographically unresolved components. In conclusion, PARAFAC analysis of the readily accessible HPLC-PDA data provides the means for unsupervised and unbiased assessment of the composition of herbal preparations, of interest for assessment of their pharmacological activity and clinical efficacy.

Coupling of a High-resolution Monoamine Oxidase-A Inhibitor Assay and HPLC–SPE–NMR for Advanced Bioactivity Profiling of Plant Extracts

INTRODUCTION Depression is a mental disease causing large personal and socio-economic problems, and new improved drugs are therefore needed. Selective monoamine oxidase A (MAO-A) inhibitors are potential anti-depressants, but discovering new MAO-A inhibitors from natural sources by bioassay-guided approaches are a lengthy and time-consuming process. New analytical technologies that allow simultaneously chemical and biological screening of extracts are therefore urgently needed. METHOD In the present study we describe coupling of a photometric microplate-based high-resolution MAO-A inhibitor assay with a hyphenated system consisting of high-performance liquid chromatography, solid-phase extraction and tube transfer nuclear magnetic resonance (HPLC-SPE-ttNMR). The standard compound clorgyline, and an extract of black pepper (Piper nigrum L.), representing a complex plant matrix, were used for proof-of-concept. RESULTS The work with clorgyline showed that the microplate-based high-resolution assay produced MAO-A inhibition profiles that easily allowed detection of submicrogram amounts of this selective MAO-A inhibitor. Furthermore, the HPLC-SPE-ttNMR/high-resolution MAO-A inhibition assay platform allowed identification of piperine and two piperine analogues as the main MAO-A inhibitors in the black pepper petroleum ether extract. CONCLUSION The HPLC-SPE-ttNMR/high-resolution MAO-A inhibition assay platform is a powerful tool for fast and efficient identification of new MAO-A inhibitors from complex extracts, and promise future advancement in the search for new anti-depressants from natural sources.