Phytochemistry (v.84, #C)

Characterization of Arabidopsis thaliana nitrile-specifier proteins (NSPs): in vitro activity of purified recombinant proteins on different glucosinolates, their dependency on Fe(II) ion and the effect of reaction conditions.Display Omitted► Heterologous expression and purification of specifier proteins. ► In vitro characterization on different glucosinolate substrates. ► Effect of ferrous ion and reaction conditions on nitrile-specifier activity.Glucosinolates are plant secondary metabolites that are part of a plant defence system against pathogens and pests, the myrosinase-glucosinolate system, in which glucosinolates get activated by enzymic degradation through thioglucoside glucohydrolases called myrosinases. Epithiospecifier protein (ESP) and nitrile-specifier proteins (NSPs) divert myrosinase-catalyzed hydrolysis of a given glucosinolate from the formation of isothiocyanate to that of epithionitrile and/or nitrile. As the biological activity of glucosinolate hydrolysis products varies considerably, a detailed characterization of these specifier proteins is of utmost importance to understand their biological role. Therefore, the Arabidopsis thaliana AtNSP1, AtNSP2 and AtNSP5 and a supposed ancestor protein AtNSP-like1 were expressed in Escherichia coli and the activity of the purified recombinant proteins was tested in vitro on three highly different glucosinolates and compared to that of purified AtESP. As previously reported, only AtESP showed epithiospecifier activity on 2-propenylglucosinolate. We further confirmed that purified AtNSP1, AtNSP2 and AtNSP5, but not the ancestor AtNSP-like1 protein, show nitrile-specifier activity on 2-propenylglucosinolate and benzylglucosinolate. We now show for the first time that in vitro AtNSP1, AtNSP2 and AtNSP5 are able to generate nitrile from indol-3-ylmethylglucosinolate. We also tested the effect of different Fe(II) ion concentrations on the nitrile-specifier activity of purified AtNSP1, AtNSP2 and AtNSP5 on 2-propenylglucosinolate and benzylglucosinolate. AtNSP-related nitrile production was highly dependent on the presence of Fe(II) ions in the reaction assay. In the absence of added Fe(II) ions nitriles were only detected when benzylglucosinolate was incubated with AtNSP1. While AtNSP1 also exhibited overall higher nitrile-specifier activity than AtNSP2 and AtNSP5 at a given Fe(II) ion concentration, the pattern of nitrile formation in relation to Fe(II) ion concentrations depended on the AtNSP and the glucosinolate substrate. The pH of the solution also affected the reaction outcome, with a higher proportion of nitrile being produced at the higher pH for AtNSP2 and AtNSP5.
Keywords: Arabidopsis thaliana; Glucosinolates; Myrosinase; Epithiospecifier protein; Nitrile-specifier proteins; Isothiocyanates; Nitriles; Fe(II) ion;

Effects of exogenous auxin and ethylene on the Arabidopsis root proteome by William O. Slade; W. Keith Ray; Patricia M. Williams; Brenda S.J. Winkel; Richard F. Helm (18-23).
A parallel comparison of auxin- and ethylene-induced protein changes in plant roots by two-dimensional gel electrophoresis and mass spectrometry indicates that these hormones elicit different effects on the most abundant proteins in Arabidopsis roots.Display Omitted► 2D-GE, MALDI TOF/TOF was used to examine auxin and ethylene effects on the root proteome. ► No overlap among the most abundant proteins affected by auxin or ethylene occurred. ► Results provide insight into auxin and ethylene regulatory networks.The phytohormones, auxin and ethylene, together control a wide range of physiological and developmental processes in plants. The lack of knowledge regarding how the underlying signaling processes are reflected at the protein level represents a major gap in understanding phytohormone signaling, including that mediated by crosstalk between auxin and ethylene. Herein is a parallel comparison of the effects of these two hormones on the Arabidopsis root proteome. Arabidopsis seedlings were exposed to 1 μm indole-3-acetic acid (IAA, auxin) or 1 μm 1-amino-cyclopropane carboxylic acid (ACC) for 24 h. Root protein extracts were fractionated using two-dimensional gel electrophoresis and the proteins that changed the most were analyzed by MALDI TOF/TOF mass spectrometry. Of the 500 total spots that were matched across all gels, 24 were significantly different after IAA exposure, while seven others were different after ACC exposure. Using rigorous criteria, identities of eight proteins regulated by IAA and five regulated by ACC were assigned. Interestingly, although both hormones affected proteins associated with fundamental cellular processes, no overlap was observed among the proteins affected by auxin or ethylene treatment. This report provides a comparison of the effects of these two hormones relative to a control utilizing equivalent treatment regimes and suggests that, while these hormones communicate to control similar physiological and transcriptional processes, they have different effects on the most abundant proteins in Arabidopsis roots.
Keywords: Arabidopsis thaliana; Cruciferae; Proteomics; Matrix-assisted laser desorption ionization time-of-flight mass spectrometry; Proteome; Root; Two-dimensional gel electrophoresis;

Myrosinases TGG1 and TGG2 from Arabidopsis thaliana contain exclusively oligomannosidic N-glycans by Eva Liebminger; Josephine Grass; Jakub Jez; Laura Neumann; Friedrich Altmann; Richard Strasser (24-30).
All N-glycosylation sites of endogenous thioglucoside glucohydrolases TGG1 and TGG2 from Arabidopsis leaves are occupied by oligomannosidic N-glycan structures, which are generated by class I α-mannosidases.Display Omitted► TGG1 and TGG2 are glycosylated with oligomannosidic N-glycans. ► TGG1 N-glycans are processed by class I alpha-mannosidases. ► TGG1 transcript levels are higher in leaves of mns1 mns2 mns3 plants.In all eukaryotes N-glycosylation is the most prevalent protein modification of secretory and membrane proteins. Although the N-glycosylation capacity and the individual steps of the N-glycan processing pathway have been well studied in the model plant Arabidopsis thaliana, little attention has been paid to the characterization of the glycosylation status of individual proteins. We report here the structural analysis of all N-glycans present on the endogenous thioglucoside glucohydrolases (myrosinases) TGG1 and TGG2 from A. thaliana. All nine glycosylation sites of TGG1 and all four glycosylation sites of TGG2 are occupied by oligomannosidic structures with Man5GlcNAc2 as the major glycoform. Analysis of the oligomannosidic isomers from wild-type plants and mannose trimming deficient mutants by liquid chromatography with porous graphitic carbon and mass spectrometry revealed that the N-glycans from both myrosinases are processed by Golgi-located α-mannosidases.
Keywords: Arabidopsis thaliana; Post-translational modification; N-Glycosylation; Glycan; Glycoprotein; Mannosidase; Thioglucoside glucohydrolase;

Biosynthesis of fomannoxin in the root rotting pathogen Heterobasidion occidentale by David Hansson; Audrius Menkis; Åke Olson; Jan Stenlid; Anders Broberg; Magnus Karlsson (31-39).
The biosynthesis of fomannoxin was investigated through a 13C-glucose labeling study. A natural product (1) was isolated and characterized, and suggested to be a key intermediate in the biosynthesis of fomannoxin.Display Omitted► The biosynthesis of fomannoxin by Heterobasidion annosum s.l. was investigated. ► 13C-labeling showed the shikimic acid pathway and MVA route to be involved. ► A prenyltransferase catalyzed alkylation was suggested to form a key intermediate.Fomannoxin is a biologically active benzohydrofuran, which has been suggested to be involved in the pathogenicity of the root rotting fungus Heterobasidion annosum sensu lato. The biosynthesis of fomannoxin was investigated through an isotopic enrichment study utilizing [1-13C]glucose as metabolic tracer. 13C NMR spectroscopic analysis revealed the labeling pattern and showed that the isoprene building block originates from the mevalonic acid pathway, whereas the aromatic motif is formed via the shikimic acid route by elimination of pyruvate from chorismic acid. A natural product, 4-hydroxy-3-(3-methylbut-2-enyl)benzaldehyde (1), was isolated and characterized, and was suggested to be a key intermediate in the biosynthesis of fomannoxin and related secondary metabolites previously identified from the H. annosum fungal species complex.
Keywords: Heterobasidion occidentale; Bondarzewiaceae; Biosynthesis investigation; Benzohydrofuran; Fomannoxin;

Functional characterization of wheat copalyl diphosphate synthases sheds light on the early evolution of labdane-related diterpenoid metabolism in the cereals by Yisheng Wu; Ke Zhou; Tomonobu Toyomasu; Chizu Sugawara; Madoka Oku; Shiho Abe; Masami Usui; Wataru Mitsuhashi; Makiko Chono; Peter M. Chandler; Reuben J. Peters (40-46).
Wheat contains multiple copalyl diphosphate synthases (CPS), whose stereochemical differentiation reflects their phylogenetic relationship with rice CPS, implying early evolution of such variation.Display Omitted► Biochemical characterization of copalyl diphosphate synthases from wheat and barley. ► Stereochemical differentiation provides rationale for expanded gene family. ► Molecular phylogenetic analysis suggests this arose early in cereal crop evolution.Two of the most agriculturally important cereal crop plants are wheat (Triticum aestivum) and rice (Oryza sativa). Rice has been shown to produce a number of diterpenoid natural products as phytoalexins and/or allelochemicals – specifically, labdane-related diterpenoids, whose biosynthesis proceeds via formation of an eponymous labdadienyl/copalyl diphosphate (CPP) intermediate (e.g., the ent-CPP of gibberellin phytohormone biosynthesis). Similar to rice, wheat encodes a number of CPP synthases (CPS), and the three CPS characterized to date (TaCPS1–3) all have been suggested to produce ent-CPP. However, several of the downstream diterpene synthases will only react with CPP intermediate of normal or syn, but not ent, stereochemistry, as described in the accompanying report. Investigation of additional CPS did not resolve this issue, as the only other functional synthase (TaCPS4) also produced ent-CPP. Chiral product characterization of all the TaCPS then established that TaCPS2 uniquely produces normal, rather than ent-, CPP, thus, providing a suitable substrate source for the downstream diterpene synthases. Notably, TaCPS2 is most homologous to the similarly stereochemically differentiated syn-CPP synthase from rice (OsCPS4), while the non-inducible TaCPS3 and TaCPS4 cluster with the rice OsCPS1 required for gibberellin phytohormone biosynthesis, as well as with a barley (Hordeum vulgare) CPS (HvCPS1) that also is characterized here as similarly producing ent-CPP. These results suggest that diversification of labdane-related diterpenoid metabolism beyond the ancestral gibberellins occurred early in cereal evolution, and included the type of stereochemical variation demonstrated here.
Keywords: Oryza sativa; Triticum aestivum; Hordeum vulgare; Graminae; Copalyl diphosphate synthase;

Functional characterization of wheat ent-kaurene(-like) synthases indicates continuing evolution of labdane-related diterpenoid metabolism in the cereals by Ke Zhou; Meimei Xu; Mollie Tiernan; Qian Xie; Tomonobu Toyomasu; Chizu Sugawara; Madoka Oku; Masami Usui; Wataru Mitsuhashi; Makiko Chono; Peter M. Chandler; Reuben J. Peters (47-55).
Wheat encodes a family of kaurene-like synthases (KSL) with varied function whose phylogenetic relationship with rice KSL implies continuing evolution.Display Omitted► Biochemical characterization of ent-kaurene(-like) synthases from wheat and barley. ► Functional diversity provides rationale for expanded gene family. ► Stereospecificity highlighted variation in upstream copalyl diphosphate synthases. ► Molecular phylogenetic analysis indicates continuing diterpenoid evolution within the cereals.Wheat (Triticum aestivum) and rice (Oryza sativa) are two of the most agriculturally important cereal crop plants. Rice is known to produce numerous diterpenoid natural products that serve as phytoalexins and/or allelochemicals. Specifically, these are labdane-related diterpenoids, derived from a characteristic labdadienyl/copalyl diphosphate (CPP), whose biosynthetic relationship to gibberellin biosynthesis is evident from the relevant expanded and functionally diverse family of ent-kaurene synthase-like (KSL) genes found in rice the (OsKSLs). Herein reported is the biochemical characterization of a similarly expansive family of KSL from wheat (the TaKSLs). In particular, beyond ent-kaurene synthases (KS), wheat also contains several biochemically diversified KSLs. These react either with the ent-CPP intermediate common to gibberellin biosynthesis or with the normal stereoisomer of CPP that also is found in wheat (as demonstrated by the accompanying paper describing the wheat CPP synthases). Comparison with a barley (Hordeum vulgare) KS indicates conservation of monocot KS, with early and continued expansion and functional diversification of KSLs in at least the small grain cereals. In addition, some of the TaKSLs that utilize normal CPP also will react with syn-CPP, echoing previous findings with the OsKSL family, with such enzymatic promiscuity/elasticity providing insight into the continuing evolution of diterpenoid metabolism in the cereal crop plant family, as well as more generally, which is discussed here.
Keywords: Wheat, Triticum aestivum; Barley, Hordeum vulgare; Rice, Oryza sativa; Graminae; Arabidopsis thaliana; Cruciferae; ent-Kaurene synthase; Phytoalexin; Phytoanticipin; Allelochemicals; Natural products biosynthesis; Plant defense;

Key structural features of cis-cinnamic acid as an allelochemical by Masato Abe; Keisuke Nishikawa; Hiroshi Fukuda; Kazunari Nakanishi; Yuta Tazawa; Tomoya Taniguchi; So-young Park; Syuntaro Hiradate; Yoshiharu Fujii; Katsuhiro Okuda; Mitsuru Shindo (56-67).
A series of cis-cinnamic acid analogues were synthesized to elucidate the key features for lettuce root growth inhibition. Inhibitory activities on elongation of lettuce roots were also evaluated.Display Omitted► cis-Cinnamic acid analogues were synthesized and their inhibitory activities evaluated on elongation of lettuce roots. ► The cis-configuration, the carboxylate and the planar ring are essential for this activity. ► The photostability of the cinnamic acid derivatives was investigated using several light sources.1-O-cis-cinnamoyl-β-d-glucopyranose is one of the most potent allelochemicals isolated from Spiraea thunbergii Sieb. It is suggested that it derives its strong inhibitory activity from cis-cinnamic acid, which is crucial for phytotoxicity. It was synthesized to confirm its structure and bioactivity, and also a series of cis-cinnamic acid analogues were prepared to elucidate the key features of cis-cinnamic acid for lettuce root growth inhibition. The cis-cyclopropyl analogue showed potent inhibitory activity while the saturated and alkyne analogues proved to be inactive, demonstrating the importance of the cis-double bond. Moreover, the aromatic ring could not be replaced with a saturated ring. However, the 1,3-dienylcyclohexene analogue showed strong activity. These results suggest that the geometry of the C–C double bond between the carboxyl group and the aromatic ring is essential for potent inhibitory activity. In addition, using several light sources, the photostability of the cinnamic acid derivatives and the role of the C–C double bond were also investigated.
Keywords: cis-Cinnamic acid; Allelochemicals; Plant growth inhibitors; Thunberg spirea; Spiraea thunbergii; Rosaceae; Lactuca sativba L.; Lettuce; Asteraceae;

On-line detection of root-induced volatiles in Brassica nigra plants infested with Delia radicum L. root fly larvae by Elena Crespo; Cornelis A. Hordijk; Rob M. de Graaf; Devasena Samudrala; Simona M. Cristescu; Frans J.M. Harren; Nicole M. van Dam (68-77).
Analysis of on-line VOC emissions by Brassica nigra roots under attack of cabbage root fly larvae, Delia radicum, using Proton-Transfer-Reaction Mass Spectrometry (PTR-MS) and Gas Chromatography–Mass Spectrometry (GC–MS).Display Omitted► We studied volatile emissions from root-herbivore infested Brassica nigra roots. ► We show that root damage induces time-resolved volatile responses in plant roots. ► Main emissions comprise sulfur containing volatiles, with early and late responses.Plants emit various volatile organic compounds (VOCs) upon herbivore attack. These VOC emissions often show temporal dynamics which may influence the behavior of natural enemies using these volatiles as cues. This study analyzes on-line VOC emissions by roots of Brassica nigra plants under attack by cabbage root fly larvae, Delia radicum. Root emitted VOCs were detected using Proton-Transfer-Reaction Mass Spectrometry (PTR-MS) and Gas Chromatography–Mass Spectrometry (GC–MS). These analyses showed that several sulfur containing compounds, such as methanethiol, dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS) and glucosinolate breakdown products, such as thiocyanates (TC) and isothiocyanates (ITC), were emitted by the roots in response to infestation. The emissions were subdivided into early responses, emerging within 1–6 h after infestation, and late responses, evolving only after 6–12 h. The marker for rapid responses was detected at m/z 60. The ion detected at m/z 60 was identified as thiocyanic acid, which is also a prominent fragment in some TC or ITC spectra. The emission of m/z 60 stopped when the larvae had pupated, which makes it an excellent indicator for actively feeding larvae. Methanethiol, DMS and DMDS levels increased much later in infested roots, indicating that activation of enzymes or genes involved in the production of these compounds may be required. Earlier studies have shown that both early and late responses can play a role in tritrophic interactions associated with Brassica species. Moreover, the identification of these root induced responses will help to design non-invasive analytical procedures to assess root infestations.
Keywords: Brassica nigra; Brassicaceae; Black mustard; Root volatile emissions; Sulfur compounds; Glucosinolate breakdown products; Isothiocyanates;

A shift from bird to beetle pollination in Protea is associated with up-regulation and change in volatile composition (non-metric multidimensional scaling below) of floral scent. Display Omitted► Pollinator shifts are often accompanied by changes in floral olfactory cues. ► Floral scents of beetle-pollinated Protea species are chemically complex and dominated by fruit esters. ► Floral scent emissions from beetle-pollinated species are 10-fold greater than those of bird-pollinated species. ► Cetoniine beetles show a significant preference for fruity Protea scents.Evolutionary shifts between pollination systems are often accompanied by modifications of floral traits, including olfactory cues. We investigated the implications of a shift from passerine bird to beetle pollination in Protea for floral scent chemistry, and also explored the functional significance of Protea scent for pollinator attraction. Using headspace sampling and gas chromatography–mass spectrometry, we found distinct differences in the emission rates and chemical composition of floral scents between eight bird- and four beetle-pollinated species. The amount of scent emitted from inflorescences of beetle-pollinated species was, on average, about 10-fold greater than that of bird-pollinated species. Floral scent of bird-pollinated species consists mainly of small amounts of “green-leaf volatiles” and benzenoid compounds, including benzaldehyde, anisole and benzyl alcohol. The floral scent of beetle-pollinated species is dominated by emissions of linalool, a wide variety of other monoterpenes and the benzenoid methyl benzoate, which imparts a fruity odour to the human nose. The number of compounds recorded in the scent of beetle-pollinated species was, on average, greater than in bird-pollinated species (45 versus 29 compounds, respectively). Choice experiments using a Y-maze showed that a primary pollinator of Protea species, the cetoniine beetle Atrichelaphinis tigrina, strongly preferred the scent of inflorescences of the beetle-pollinated Protea simplex over those of the bird-pollinated sympatric congener, Protea roupelliae. This study shows that a shift from passerine bird- to insect-pollination can be associated with marked up-regulation and compositional changes in floral scent emissions.
Keywords: Protea; Proteaceae; Atrichelaphinis tigrina; Cetoniinae; Gas chromatography–mass spectrometry; Beetle pollination; Pollination shift;

Glucomannan and branched (1 → 3)(1 → 6) β-glucan from the aposymbiotically grown Physcia kalbii mycobiont by Lucimara M.C. Cordeiro; Vanessa de Fátima Reinhardt; Marcello Iacomini (88-93).
The glucan consisted of a (1 → 3)-linked glucopyranosyl backbone branched at C-6 by 6-O- and 2,6-O-linked-Glcp units. The glucomannan had (1 → 6)-linked α-Manp units in the main chain, almost all being substituted at O-2 by α-Manp and α-Glcp units.Display Omitted► The mycobiont of Physcia kalbii was aposymbiotically cultivated. ► The mycobiont has a branched β-(1 → 3),(1 → 6)-glucan. ► The mycobiont has a glucomannan, which could be typical of the Physciaceae.Cultures of the mycobiont Physcia kalbii were obtained from germinated ascospores and cultivated on Sabouraud-Sucrose-agar medium. Alkaline extraction of freeze-dried mycelia provided a branched (1 → 3),(1 → 6)-β-glucan and a glucomannan, whose chemical structure was determined by monosaccharide composition, methylation, controlled Smith degradation and NMR spectroscopic analysis. The β-glucan had a (1 → 3)-linked β-glucopyranosyl backbone, partially substituted (approx. 50% of the units) at O-6. The side chains were formed by 6-O- (∼82%) and 2,6-O-linked-β-Glcp units, while the non-reducing ends were formed by β-glucopyranosyl residues. The glucomannan had (1 → 6)-linked α-Manp units in the main chain, almost all being substituted at O-2 by α-Manp and α-Glcp units. This glucomannan could be a typical polysaccharide of lichens from the family Physciaceae.
Keywords: Lichen; Physcia; Mycobiont; Polysaccharides; Glucomannan; Branched β-glucan;

Vibrational spectroscopy and GC–MS data analysed with MetAlign software, have been used to investigate the potential of two Salvia species indigenous to South Africa as sources of α-bisabolol, a commercially important aroma chemical.Display Omitted► Chemotaxonomic differentiation of S. stenophylla and S. runcinata was achieved. ► Chemotypes of S. stenophylla and S. runcinata rich in α-bisabolol were identified. ► PLS calibration models were able to predict α-bisabolol content.α-Bisabolol is a commercially important aroma chemical currently obtained from the Candeia tree (Vanillosmopsis erythropappa). Continuous unsustainable harvesting of the Candeia tree has prompted the urgent need to identify alternative crops as a source of this commercially important sesquiterpene alcohol. A chemotaxonomic assessment of two Salvia species indigenous to South Africa is presented and recommended as a potential source of α-bisabolol. The essential oil obtained by hydrodistillation of the aerial parts was analysed by gas chromatography coupled to mass spectrometry (GC–MS) and mid-infrared spectroscopy (MIRS). Orthogonal projections to latent structures–discriminant analysis (OPLS–DA) were used for multivariate classification of the oils based on GC–MS and MIRS data. Partial least squares (PLS) calibration models were developed on the MIRS data for the quantification of α-bisabolol using GC–MS as the reference method. A clear distinction between Salvia stenophylla and Salvia runcinata oils was observed using OPLS–DA on both GC–MS and MIRS data. The MIR calibration model showed high coefficient of determination (R 2  = 0.999) and low error of prediction (RMSEP = 0.540%) for α-bisabolol content.
Keywords: Salvia stenophylla; Salvia runcinata; α-Bisabolol; MetAlign; Mid-infrared spectroscopy; Chemometrics;

6-OHDA is auto-oxidized rapidly by molecular oxygen to form the corresponding p-quinone and H2O2. Both H2O2 and p-quinone induce apoptosis are inhibited by the natural flavonoid, nephrocizin. The latter can form a nephrocizin dimer (5′,5′-conjugate) to diminish intracellular hydroxyl radical (OH) levels produced by H2O2..Display Omitted► Neuroprotective effects of nephrocizin in 6-OHDA-induced PC12 cells was evaluated. ► Nephrocizin decreased caspase-3 and -8 activities induced by H2O2 or p-quinone. ► Nephrocizin scavenged OH through forming 5′,5′-nephrocizin dimers. ► These chemical and biological studies suggested potential screening methods.The neurotoxin 6-hydroxydopamine (6-OHDA) has been widely used to generate an experimental model of Parkinson’s disease. This model is crucial in the search for compounds that diminish 6-OHDA-induced nerve growth factor (NGF)-differentiated PC12 cell death. Nephrocizin (luteolin-7-O-β-d-glucopyranoside), a flavone glycoside, was isolated from widely distributed plants. The protective effects of pre-treatment with nephrocizin on the induced neurotoxicity in PC12 cells by 6-OHDA and its oxidative products, H2O2 and p-quinone, were evaluated herein. Nephrocizin promoted cell viability, scavenged ROS-related products, increased cellular glutathione (GSH) levels, and reduced caspase-3 and -8 activities in 6-OHDA-, H2O2-, or p-quinone-treated PC12 cells. Furthermore, nephrocizin-conjugated metabolites in PC12 cells were identified with the boronate-affinity method and LC-MS technology, and preferential regioselectivity at the C2′ and C5′ positions by the nephrocizin-GSH (or NAC) adduct method was observed. These lines of evidence established that nephrocizin could form a dimer to diminish the intracellular ROS. These results demonstrate the first neuroprotective mechanism of nephrocizin against 6-OHDA-, H2O2- or p-quinone-induced cytotoxicity in PC12 cells via chemical and biological studies. These dietary antioxidants are potential candidates for use in intervention in neurodegenerative diseases.
Keywords: Formosa date palm; Phoenix hanceana Naudin var. formosana; Arecaceae; 6-Hydroxydopamine; H2O2; p-Quinone; Neuroprotection; Nephrocizin; Chemical and biological studies;

Studies of naturally occurring friedelane triterpenoids as insulin sensitizers in the treatment type 2 diabetes mellitus by Alejandro E. Ardiles; Águeda González-Rodríguez; Marvin J. Núñez; Nayra R. Perestelo; Virginia Pardo; Ignacio A. Jiménez; Ángela M. Valverde; Isabel L. Bazzocchi (116-124).
Nine fridelane triterpenes and twenty two known ones were isolated from Celastraceae species. Two compounds exhibited potent insulinomimetic effect on insulin signaling in hepatocytes..Display Omitted► A series of friedelane-type triterpenes were isolated from Celastraceae species. ► Two friedelanes directly activated IR tyrosine phosphorylation in hepatocytes. ► They exhibited increased insulin-mediated Akt signalling in hepatocytes. ► Friedelane-type triterpenes have potential therapeutic use in T2DM.Type 2 diabetes mellitus (T2DM) is a rapidly expanding public epidemic and frequently results in severe vascular complications. In an attempt to find anti-diabetic agents, we report herein on the isolation, structural elucidation and bioactivity of nine friedelane-type triterpenes (19) and twenty two known ones (1031) from the root barks of Celastrus vulcanicola and Maytenus jelskii. Their structures were elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques. Two compounds from this series (1 and 3) exhibited increased insulin-mediated signalling, which suggests these friedelane triterpenes have potential therapeutic use in insulin resistant states.
Keywords: Celastraceae; Celastrus vulcanicola; Maytenus jelskii; Friedelane triterpenes; Type 2 diabetes mellitus; Insulin receptor; Receptor tyrosine phosphorylation; Anti-diabetic agents;

Oleanane saponins from Bellis sylvestris Cyr. and evaluation of their phytotoxicity on Aegilops geniculata Roth by Monica Scognamiglio; Brigida D’Abrosca; Vittorio Fiumano; Angela Chambery; Valeria Severino; Nikolaos Tsafantakis; Severina Pacifico; Assunta Esposito; Antonio Fiorentino (125-134).
Six phytotoxic saponins, Besylvosides I–VI, have been isolated from the leaves of Bellis sylvestris..Display Omitted► Six novel oleanane saponins, Besylvosides I–VI, were isolated. ► Structures have been elucidated by 2D NMR and ESI Q-TOF MS2 spectrometry. ► Their phytotoxicity has been assessed against the species Aegilops geniculata. ► Saponins strongly inhibited the shoot elongation at the highest concentration.Six oleanane saponins were isolated for the first time from leaves of Bellis sylvestris Cyr., the southern daisy. Their structures were established by the extensive use of 2D-NMR experiments, including COSY, TOCSY, NOESY, HSQC, HMBC, CIGAR, H2BC, and HSQC–TOCSY, along with Q-TOF HRMS2 analysis. All of the compounds are constituted by bayogenin as aglycone, and characterized by the presence of an oligosaccharide moiety, consisting of two to four sugar unities esterified at the C-28 carboxyl carbon. One of the isolated compounds is a bisdesmoside containing an additional sugar moiety at the C-3 carbon.The phytotoxic activity assayed against Aegilops geniculata Roth., a coexisting test species, has been evaluated revealing that all the compounds, at the highest concentrations, showed strong phytotoxicity against the leaf development.
Keywords: Bellis sylvestris Cyr.; Asteraceae; Saponins; Besylvosides; Spectroscopic analysis; Aegylops geniculata Roth.; Phytotoxicity;

Biotransformation of natural compounds: Unexpected thio conjugation of Sch-642305 with 3-mercaptolactate catalyzed by Aspergillus niger ATCC 16404 cells by Emilie Adelin; Marie-Thérèse Martin; Marie-Françoise Bricot; Sylvie Cortial; Pascal Retailleau; Jamal Ouazzani (135-140).
Sch-642305 is converted with Aspergillus niger ATCC 16404 resting cells to two unexpected thio-conjugates, compounds (1) and (2). The biological significance as well as the reaction mechanisms are discussed.Display Omitted► We investigate the bioconversion of the natural compounds Sch-642305. ► We isolate two unexpected thio-conjugation derivatives, difficult to access through chemical synthesis. ► We evaluate the antibiotic and cytotoxic activities of these compounds. ► We propose the reaction mechanisms involved.Sch-642305 is produced by the endophytic fungi Phomopsis sp. CMU-LMA and exhibits both antimicrobial and cytotoxic activities. The incubation of Sch-642305 with Aspergillus niger ATCC 16404 resting cells leads to two unexpected thio conjugates. Compound (1) is formed by the addition of the cysteine metabolite 3-mercaptolactate to the double bond of Sch-642305. Compound (1) undergoes an intramolecular rearrangement to give compound (2), which contains two rings: a five-membered hydroxylactone ring and a five-membered thiophene ring. The absolute configuration of compound (1) is similar to that of the parent compound, but the configuration of the mercaptolactate side-chain was not determined. The absolute configuration of compound (2) was deduced from the crystal structure and confirmed by the anomal effect of the sulfur atom. To the best of our knowledge, this is the first time such a conjugation rearrangement reactions were observed. The biological significance and the reaction mechanisms are discussed. Compound (1) exhibits a weak antimicrobial activity against Gram-positive bacteria, whereas derivatives (1) and (2) showed an IC50 of 1 and 1.2 μM, respectively, against colonic epithelial cancer cells.
Keywords: Sch-642305; Biotransformation; Aspergillus niger; Thio conjugation; Chemodiversity;

Rare, seven-membered cyclic ether labdane diterpenoid from Dodonaea polyandra by Bradley S. Simpson; David J. Claudie; Nick M. Smith; Ross A. McKinnon; Susan J. Semple (141-146).
As part of ongoing research of Dodonaea polyandra, an individual plant displaying male flowers was investigated. This yielded four labdane diterpenoids, of which one contains a rare seven membered cyclic ether ring, the first isolated from a terrestrial source.Display Omitted► First phytochemical investigation of a male individual of Dodonaea polyandra. ► Labdane diterpenoids are now shown to exist in this species. ► A rare seven-membered cyclic ether labdane diterpenoid, is reported.Previous phytochemical studies on the leaf resin of dioecious plant species Dodonaea polyandra have identified the presence of furanoclerodane diterpenoids. As part of ongoing research on this species the chemical profile of an individual plant displaying male flowers was investigated. Repeated chromatographic separation of a resinous extract from the leaves of the plant yielded three labdane diterpenoids, 13,17-epoxy-13-methyl-15-oxo-labda-7-ene (1), 17-hydroxy-13-methyl-labda-7,13Z-diene-15-oic acid (2) and 13-methyl-17-oxo-labda-7,13Z-diene-15-oic acid (3) and a fourth known labdane diterpenoid (4) reported as being isolated from a natural source for the first time. Structural elucidation was carried out using conventional 1D and 2D NMR and mass spectrometry together with other complementary techniques (UV and IR). The leaf extract from this individual of D. polyandra with male flowers present displays a marked difference in the chemical composition of diterpenoids compared to previously studied extracts from the leaves of this species.
Keywords: Dodonaea polyandra; Diterpenoid; Labdane;

Saponins from Astragalus hareftae (NAB.) SIRJ. by Ibrahim Horo; Erdal Bedir; Milena Masullo; Sonia Piacente; Fevzi Özgökçe; Özgen Alankuş-Çalışkan (147-153).
Five saponin glycosides, together with 15 known compounds were isolated from the roots of Astragalus hareftae. Compounds 3 and 4 are monosaccharidic cycloartane-type triterpenoids which are rarely encountered in nature.Display Omitted► A phytochemical study was performed on Astragalus hareftae. ► Four cycloartane-type triterpene glycosides were isolated. ► A soyasapogenol glycoside was isolated. ► Two monosaccharidic cycloartane-type triterpenoids were elucidated, uncommon in nature.Four cycloartane- (hareftosides A–D) and oleanane-type triterpenoids (hareftoside E) were isolated from Astragalus hareftae along with fifteen known compounds. Structures of the compounds were established as 3,6-di-O-β-d-xylopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane (1), 3,6,24-tri-O-β-d-xylopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane (2), 3-O-β-d-xylopyranosyl-3β,6α,16β,25-tetrahydroxy-20(R),25(S)-epoxycycloartane (3), 16-O-β-d-glucopyranosyl-3β,6α,16β,25-tetrahydroxy-20(R),24(S)-epoxycycloartane (4), 3-O-[β-d-xylopyranosyl-(1→2)-O-β-d-glucopyranosyl-(1→2)-O-β-d-glucuronopyranosyl]-soyasapogenol B (5) by the extensive use of 1D- and 2D-NMR experiments along with ESI-MS and HR-MS analyses.
Keywords: Astragalus; Saponin; Cycloartane; Oleanane; Hareftoside;

Terpenoids from Russula lepida and R. amarissima (Basidiomycota, Russulaceae) by Marco Clericuzio; Claudio Cassino; Federica Corana; Giovanni Vidari (154-159).
Aristolane sesquiterpenes and seco-cucurbitane triterpenes were isolated from sporocarps of Russula lepida and R. amarissima (Basidiomycota). Cucurbitanes as 14 (showing cell-growth inhibitor activity) may have a protection role against parasites.Display Omitted► Four aristolane sesquiterpenes and one seco-cucurbitane triterpene were isolated. ► A 2S rather than 2R configuration was assigned to known 2-hydroxyaristolone. ► No enzymatic transformation occurred in injured fruiting bodies (after 20′ at rt). ► Compound 14 was active in a cell growth inhibitory bioassay.Four aristolane sesquiterpenes were isolated from the fruiting bodies of Russula lepida and R. amarissima, namely (1R,2S)-1,2-dihydroxyaristolone (6), (2S,11S)-2,12-dihydroxy-aristolone (7), (1R,2S,11S)-1,2,12-trihydroxyaristolone (8), (1S,2S,11S)-1,2,12-trihydroxy-aristolone (9). In addition, a seco-cucurbitane triterpene, i.e. 3,4-secocucurbita-4,24E-diene-3-hydroxy-26-carboxylic acid (14) was isolated from both species. The configuration at C-2 of the already known rulepidol (2-hydroxyaristolone, 5) was corrected as S instead of R. Several more aristolane and nardosinane sesquiterpenes, as well as cucurbitane triterpenes, already reported both from European and Chinese samples of R. lepida, were also isolated. Compound 14 showed moderate cell growth inhibitory activity.
Keywords: Basidiomycota; Russula lepida; Russula amarissima; Sesquiterpenes; Aristolane; Triterpenes; Cucurbitane; Cell growth inhibitory activity;

Antiviral chlorinated daphnane diterpenoid orthoesters from the bark and wood of Trigonostemon cherrieri by Pierre-Marie Allard; Pieter Leyssen; Marie-Thérèse Martin; Mélanie Bourjot; Vincent Dumontet; Cécilia Eydoux; Jean-Claude Guillemot; Bruno Canard; Cyril Poullain; Françoise Guéritte; Marc Litaudon (160-168).
The chemical investigation of the bark and wood of Trigonostemon cherrieri (Euphorbiaceae) led to the isolation of ten chlorinated daphnane diterpenoid orthoesters (DDO) named trigocherrins A–F (16) and trigocherriolides A–D (710). Several DDO exhibited strong antiviral activity against chinkungunya, Sindbis and Semliki forest virus.Display Omitted► Ten daphnane diterpenoid orthoesters were isolated from Trigonostemon cherrieri. ► They all possess a mono- or dichlorovinyl group on the daphnane core. ► They were isolated using bioassay-guided and LC/MS-guided fractionations. ► They are a class of non-nucleosidic inhibitor of the DENV NS5 RdRp. ► They showed strong antiviral activity against CHIK, Sindbis and Semliki forest virus.The chemical study of the bark and the wood of Trigonostemon cherrieri, a rare endemic plant of New Caledonia, led to the isolation of a series of highly oxygenated daphnane diterpenoid orthoesters (DDO) bearing an uncommon chlorinated moiety: trigocherrins A–F and trigocherriolides A–D. Herein, we describe the isolation and structure elucidation of the DDO (trigocherrins B–F and trigocherriolides A–D). We also report the antiviral activity of trigocherrins A, B and F (1, 2 and 6) and trigocherriolides A, B and C (79) against various emerging pathogens: chikungunya virus (CHIKV), Sindbis virus (SINV), Semliki forest virus (SFV) and dengue virus (DENV).
Keywords: Trigonostemon cherrieri; Euphorbiaceae; Trigocherrin; Trigocherriolide; Daphnane; Chlorinated diterpenes; Antiviral; Chikungunya virus; Sindbis virus; Semliki forest virus; Dengue virus;

Elucidation of the biosynthesis of the di-C-glycosylflavone isoschaftoside, an allelopathic component from Desmodium spp. that inhibits Striga spp. development by Mary L. Hamilton; Serge P. Kuate; Melissa Brazier-Hicks; John C. Caulfield; Ruth Rose; Robert Edwards; Baldwyn Torto; John A. Pickett; Antony M. Hooper (169-176).
A di-C-glycosyl-2-hydroxynaringenin is biosynthesised from 2-hydroxynaringenin in Desmodium uncinatum and Desmodium intortum by sequential glycosylation with UDP-glucose and UDP-arabinose. Chemical dehydration is required implying a dehydratase enzyme has a role in vivo to complete the biosynthesis.Display Omitted► Di-C-glycosylflavones are biosynthesised by sequential glycosylation of 2-hydroxynaringenin. ► UDP-glucose and UDP-arabinose are the sugar donors. ► A dehydratase is required to fully dehydrate the di-C-glycosylated flavonoid intermediate. ► Specific C-glycosyltransferases are required for each glycosylation.Isoschaftoside, an allelopathic di-C-glycosylflavone from Desmodium spp. root exudates, is biosynthesised through sequential glucosylation and arabinosylation of 2-hydroxynaringenin with UDP-glucose and UDP-arabinose. Complete conversion to the flavone requires chemical dehydration implying a dehydratase enzyme has a role in vivo to complete the biosynthesis. The C-glucosyltransferase has been partially characterised and its activity demonstrated in highly purified fractions.
Keywords: Desmodium uncinatum; Desmodium intortum; Biosynthesis; Di-C-glycosylflavone; C-Glycosyltransferase; Isoschaftoside;

Lanostane triterpenoids and sterols from Antrodia camphorata by Hui-Chi Huang; Chih-Chuang Liaw; Hsin-Ling Yang; You-Cheng Hseu; Hsiou-Ting Kuo; Yao-Ching Tsai; Shih-Chang Chien; Sakae Amagaya; Yu-Chang Chen; Yueh-Hsiung Kuo (177-183).
Lanostane triterpenes (14) and sterols (56) were isolated from the fruiting bodies and mycelia of solid culture of Antrodia camphorata together with seven known compounds. The structures were determined by 1D- and 2D-NMR techniques, and mass spectrometry.Display Omitted► Six previously-unreported compounds were isolated from Antrodia camphorata. ► The structures of four lanostane triterpenes and two sterols were elucidated by spectroscopic methods. ► The absolute configuration of a sterol was assigned by the Mosher ester method.Four lanostane triterpenes, 3,7,11-trioxo-5α-lanosta-8,24(E)-dien-26-oic acid, methyl 11α-3,7-dioxo-5α-lanosta-8,24(E)-dien-26-oate, methyl 3,7,11,12,15,23-hexaoxo-5α-lanost-8-en-26-oate, and ethyl 3,7,11,12,15,23-hexaoxo-5α-lanost-8-en-26-oate, two sterols, (14α,22E)-14-hydroxyergosta-7,22-diene-3,6-dione and a steroid named as camphosterol A were isolated from a mixture of fruiting bodies and mycelia of solid cultures of Antrodia camphorata. The 1H and 13C NMR spectra of all compounds were fully assigned using a combination of 2D NMR experiments, including COSY, HMQC, HMBC and NOESY sequences. Six compounds were evaluated for cytotoxicity against several human tumor cell lines, all of which has moderate activity.
Keywords: Antrodia camphorata; Polyporales; Fomitopsidaceae; Cinnamomun kanehirai Hay; Lauraceae; Chinese herb; Lanostane-type triterpenoids; Fungal sterols; Camphosterol A;