Phytochemistry (v.77, #C)

Oleanolic acid by Jacob Pollier; Alain Goossens (10-15).
Oleanolic acid (3β-hydroxyolean-12-en-28-oic acid) is a pentacyclic triterpenoid compound with various pharmacological activities and a widespread occurrence throughout the plant kingdom.Display Omitted► Oleanolic acid is a triterpenoid compound present in many plant species. ► The complete biosynthetic pathway leading to oleanolic acid is elucidated. ► Oleanolic acid exerts pharmacological activities such as hepatoprotective effects. ► Chemical derivatives of oleanolic acid have increased pharmacological activities. ► Perspectives on heterologous biosynthesis of oleanolic acid and its derivatives.Oleanolic acid (3β-hydroxyolean-12-en-28-oic acid) is a pentacyclic triterpenoid compound with a widespread occurrence throughout the plant kingdom. In nature, the compound exists either as a free acid or as an aglycone precursor for triterpenoid saponins, in which it can be linked to one or more sugar chains. Oleanolic acid and its derivatives possess several promising pharmacological activities, such as hepatoprotective effects, and anti-inflammatory, antioxidant, or anticancer activities. With the recent elucidation of its biosynthesis and the imminent commercialization of the first oleanolic acid-derived drug, the compound promises to remain important for various studies. In this review, the recent progress in understanding the oleanolic acid biosynthesis and its pharmacology are discussed. Furthermore, the importance and potential application of synthetic oleanolic acid derivatives are highlighted, and research perspectives on oleanolic acid are given.
Keywords: Oleanolic acid; CDDO-Me; Bardoxolone methyl; GC–MS; Nrf2; Synthetic triterpenes; Saponins; Hepatoprotective; Anti-inflammatory; Anticancer;

Glucosinolate structures in evolution by Niels Agerbirk; Carl Erik Olsen (16-45).
We critically review documented glucosinolate structures, methods for qualitative glucosinolate analysis, glucosinolate evolution, and recent advances in glucosinolate biochemistry. Rather than being neutral markers of evolutionary origin, glucosinolates are bioactive specialized metabolites, and their evolution can be traced using phylogenetic trees.Display Omitted► We provide a critical review of reported natural glucosinolate structures. ► A considerable number of suggested structures are insufficiently documented. ► By 2011, around 132 natural glucosinolates were scientifically documented. ► Glucosinolates are specialized metabolites subject to frequent evolution. ► Side chain specific in planta metabolic reactions may influence glucosinolate evolution.By 2000, around 106 natural glucosinolates (GSLs) were probably documented. In the past decade, 26 additional natural GSL structures have been elucidated and documented. Hence, the total number of documented GSLs from nature by 2011 can be estimated to around 132. A considerable number of additional suggested structures are concluded not to be sufficiently documented. In many cases, NMR spectroscopy would have provided the missing structural information. Of the GSLs documented in the past decade, several are of previously unexpected structures and occur at considerable levels. Most originate from just four species: Barbarea vulgaris, Arabidopsis thaliana, Eruca sativa and Isatis tinctoria. Acyl derivatives of known GSLs comprised 15 of the 26 newly documented structures, while the remaining exhibited new substitution patterns or chain length, or contained a mercapto group or related thio-functionality.GSL identification methods are reviewed, and the importance of using authentic references and structure-sensitive detection methods such as MS and NMR is stressed, especially when species with relatively unknown chemistry are analyzed. An example of qualitative GSL analysis is presented with experimental details (group separation and HPLC of both intact and desulfated GSLs, detection and structure determination by UV, MS, NMR and susceptibility to myrosinase) with emphasis on the use of NMR for structure elucidation of even minor GSLs and GSL hydrolysis products. The example includes identification of a novel GSL, (R)-2-hydroxy-2-(3-hydroxyphenyl)ethylglucosinolate.Recent investigations of GSL evolution, based on investigations of species with well established phylogeny, are reviewed. From the relatively few such investigations, it is already clear that GSL profiles are regularly subject to evolution. This result is compatible with natural selection for specific GSL side chains. The probable existence of structure-specific GSL catabolism in intact plants suggests that biochemical evolution of GSLs has more complex implications than the mere liberation of a different hydrolysis product upon tissue disruption.
Keywords: Critical review; Documented glucosinolate structures; Qualitative analysis; Structure elucidation; Brassicales phylogeny; Evolution;

Actinidin, a protease from kiwifruit, induces changes in morphology and adhesion of T84 intestinal epithelial cells by Milena Čavić; Milica Grozdanović; Aleksandar Bajić; Tatjana Srdić-Rajić; Pavle R. Anđjus; Marija Gavrović-Jankulović (46-52).
Kiwifruit allergen actinidin induces protease-dependent morphology changes of T84 intestinal cells, leading to cell rounding and desquamation of the epithelial monolayer, without affecting cell viability.Display Omitted► Actinidin is a cysteine protease from kiwifruit with allergenic properties. ► We investigated if actinidin induces changes in morphology and adhesion of T84 cells. ► Actinidin led to cell rounding and desquamation of the T84 epithelial monolayer. ► The observed effects were protease-, time- and concentration-dependant. ► Viability of T84 epithelial cells was not compromised upon actinidin treatment.Actinidin belongs to the papain-like family of cysteine proteases and is a major kiwifruit allergen. In this study, the effect of actinidin on cellular morphology and adhesion of T84 intestinal cells was investigated. Both rounding and detachment of T84 cells were observed upon actinidin treatment. The morphological changes and cell desquamation was protease-dependent, as well as time- and concentration-dependent. Changes of intercellular adhesion and adhesion of epithelial cells to collagen upon actinidin treatment could be responsible for the cell rounding and give rise to discontinuous breaches in the epithelial monolayer observed in this study. Actinidin’s action on cell morphology, adhesion and monolayer integrity were not due to compromised viability of T84 epithelial cells, as confirmed by MTT assay and flow cytometric analysis of the cell cycle. Damage to the epithelial monolayer of the intestine induced by actinidin should be further evaluated as an important factor in the development of kiwifruit allergy and other intestinal disorders.
Keywords: Actinidia sp.; Kiwifruit; Actinidin; T84 cells; Intestinal disorders;

Vein Patterning 1-encoded progesterone 5β-reductase: Activity-guided improvement of catalytic efficiency by Peter Bauer; Kristin Rudolph; Frieder Müller-Uri; Wolfgang Kreis (53-59).
Vein Patterning 1-encoded progesterone 5β-reductases (P5βR) are substrate-promiscuous enone reductases. Amino acid residues responsible for P5βR ‘weakness’ were identified and the catalytic efficiency of P5βR of D. lanata was improved by site-directed mutagenesis.Display Omitted► Vein Patterning 1-encoded progesterone 5β-reductases are substrate-promiscuous enone reductases. ► Catalytically ‘strong’ and ‘weak’ progesterone 5β-reductases were identified. ► An activity-guided screen was used to propose ‘hot spots’ for site-directed mutagenesis. ► Catalytic efficiency of Vein Patterning 1-encoded progesterone 5β-reductase was improved. ► Amino acid residues responsible for progesterone 5β-reductase ‘weakness’ were substituted.Progesterone 5β-reductases (P5βR; EC 1.3.99.6) encoded by Vein Patterning 1 (VEP1) genes are capable of reducing the C=C double-bond of a variety of enones enantioselectively. Sequence and activity data of orthologous P5βRs were used to define a set of residues possibly responsible for the large differences in enzyme activity seen between rAtSt5βR and rDlP5βR, recombinant forms of P5βRs from Arabidopsis thaliana and Digitalis lanata, respectively. Tyrosine-156, asparagine-205 and serine-248 were identified as hot spots in the rDlP5βR responsible for its low catalytic efficiency. These positions were individually substituted for amino acids found in the strong rAtSt5βR in the corresponding sites. Kinetic constants were determined for rDlP5βR and its mutants as well as for rAtSt5βR using progesterone and 2-cyclohexen-1-one as substrates. Enzyme mutants in which asparagine-205 was substituted for methionine or alanine showed considerably lower km and higher K cat/k m values than the wild-type DlP5βR, approaching the catalytic efficiency of strong P5βRs. The introduced mutations not only lead to an improved capability to reduce progesterone but also to altered substrate preference. Our findings provided structural insights into the differences seen among the natural P5βRs with regard to their substrate preferences and catalytic efficiencies.
Keywords: Digitalis lanata; Plantaginaceae; Enone reductase; Site-directed mutagenesis; Rational protein engineering;

Benzophenone synthase from Garcinia mangostana L. pericarps by Natsajee Nualkaew; Hiroyuki Morita; Yoshihiko Shimokawa; Keishi Kinjo; Tetsuo Kushiro; Wanchai De-Eknamkul; Yutaka Ebizuka; Ikuro Abe (60-69).
The cDNA of a benzophenone synthase from Garcinia mangostana L. (GmBPS) was cloned, expressed, and mutated. The recombinant GmBPS was able to utilise various substrates and 1–3 molecules of malonyl CoA to form a variety of phloroglucinol and polyketide-lactone products.Display Omitted► Benzophenone synthase (GmBPS) was cloned from Garcinia mangostana. ► GmBPS utilised various substrates and 1–3 molecules of malonyl-CoA. ► The enzymatic products included phloroglucinol and polyketide-lactone compounds. ► The total volume of GmBPS active site is smaller than that of chalcone synthase. ► Site-directed mutation to accommodate larger aromatic substrates was performed.The cDNA of a benzophenone synthase (BPS), a type III polyketide synthase (PKS), was cloned and the recombinant protein expressed from the fruit pericarps of Garcinia mangostana L., which contains mainly prenylated xanthones. The obtained GmBPS showed an amino acid sequence identity of 77–78% with other plant BPSs belonging to the same family (Clusiaceae). The recombinant enzyme produced 2,4,6-trihydroxybenzophenone as the predominant product with benzoyl CoA as substrate. It also accepted other substrates, such as other plant PKSs, and used 1–3 molecules of malonyl CoA to form various phloroglucinol-type and polyketide lactone-type compounds. Thus, providing GmBPS with various substrates in vivo might redirect the xanthone biosynthetic pathway.
Keywords: Garcinia mangostana; Clusiaceae; Xanthones; Benzophenone synthase; Polyketide synthase;

Three lipoxygenase genes were identified from Marchantia polymorpha. They preferentially use eicosapentaenoic acid as a substrate to produce 15S-HPEPE or 11S-HPEPE.Display Omitted► Three genes homologous to plant lipoxygenase were identified from Marchantia polymorpha. ► MpLOX1 showed 11S- and 15S-LOX activities for EPA substrate and 15S-LOX activity for AA. ► MpLOX2 and MpLOX3 exhibited 15S-LOX activity for both EPA and AA.Three genes homologous to plant lipoxygenase genes were identified from the EST libraries of Marchantia polymorpha, in order to clarify the function of LOXs in bryophytes. Full-length genes were isolated using 5′- and 3′-RACE methods and named MpLOX1, MpLOX2, and MpLOX3, respectively. To investigate the enzymatic activities of liverwort LOXs, recombinant MpLOX1, MpLOX2, and MpLOX3 proteins were prepared from Escherichia coli cells expressing the corresponding gene. LC–MS/MS analyses and chiral column chromatography of their reaction products showed that MpLOX1 codes for 11S/15S-lipoxygenase against eicosapentaenoic acid and for 15S-lipoxygenase against arachidonic acid, and that MpLOX2 and MpLOX3 code for 15S-lipoxygenase against eicosapentaenoic and arachidonic acids. Phylogenetic analysis showed that the liverwort lipoxygenase genes separated from the ancestor of higher plants in the early stages of plant evolution. Quantification analyses suggested that arachidonic acid and eicosapentaenoic acid were preferred substrates. Furthermore, each liverwort lipoxygenase exhibited highest activity at pH 7.0 and dependency on Ca2+ ion in the oxygenation reaction.
Keywords: Marchantia polymorpha; Bryophyte; Lipoxygenase; Oxylipin; Eicosanoid; Arachidonic acid; Eicosapentaenoic acid;

Integration of expressed sequence tag and targeted metabolite profile databases for cell cultures of 18 plant species provides a repository of candidate biosynthetic genes involved in benzylisoquinoline alkaloid metabolism.Display Omitted► We established EST and metabolomics resources for 18 alkaloid-accumulating cell cultures. ► Our EST libraries contain ∼3500 unigenes per species for a total of 58,787 unigenes. ► More than 250 putative biosynthetic gene candidates were identified with potential functions. ► Mass spectrometry was used to identify and quantify 72 alkaloids. ► Metabolite profiles provide a rational basis predicting the function of novel enzymes.Benzylisoquinoline alkaloids (BIAs) are a large and diverse group of ∼2500 specialized metabolites found predominantly in plants of the order Ranunculales. Research focused on BIA metabolism in a restricted number of plant species has identified many enzymes and cognate genes involved in the biosynthesis of compounds such as morphine, sanguinarine and berberine. However, the formation of most BIAs remains uncharacterized at the molecular biochemical level. Herein a compendium of sequence- and metabolite-profiling resources from 18 species of BIA-accumulating cell cultures was established, representing four related plant families. Our integrated approach consisted of the construction of EST libraries each containing approximately 3500 unigenes per species for a total of 58,787 unigenes. The EST libraries were manually triaged using known BIA-biosynthetic genes as queries to identify putative homologs with similar or potentially different functions. Sequence resources were analyzed in the context of the targeted metabolite profiles obtained for each cell culture using electrospray-ionization and collision-induced dissociation mass spectrometry. Fragmentation analysis was used for the identification or structural characterization coupled with the relative quantification of 72 BIAs, which establishes a key resource for future work on alkaloid biosynthesis. The metabolite profile obtained for each species provides a rational basis for the prediction of enzyme function in BIA metabolism. The metabolic frameworks assembled through the integration of transcript and metabolite profiles allow a comparison of BIA metabolism across several plant species and families. Taken together, these data represent an important tool for the discovery of BIA biosynthetic genes.
Keywords: Ranunculales; Benzylisoquinoline alkaloids; Transcriptomics; Targeted metabolite profiling; Plant cell culture; Gene discovery;

Metabolism of terpenes in the response of grape (Vitis vinifera L.) leaf tissues to UV-B radiation by Mariana Gil; Mariela Pontin; Federico Berli; Rubén Bottini; Patricia Piccoli (89-98).
Grape responds to low intensity UV-B by increasing membrane-related triterpenes (1) suggesting acclimation, while under high UV-B treatment they accumulated terpenes with antioxidant properties (2), and the stress-hormone ABA (3), suggesting mechanisms of defense.Display Omitted► We investigated terpenes in grapevines exposed to UV at low and high intensities. ► Low UV-B augmented membrane triterpenes suggesting a mechanism of acclimation. ► High UV-B increased antioxidant terpenes and ABA suggesting mechanism of defense.This study investigated the terpene profiles as determined by GC–EIMS analysis of in vitro cultured plants of Vitis vinifera exposed to a “field-like” dose of UV-B (4.75 kJ m−2  d−1) administered at two different fluence rates (low, 16 h at 8.25 μW cm−2, and high 4 h at 33 μW cm−2). Low UV-B treatment increased levels of the membrane-related triterpenes sitosterol, stigmasterol and lupeol, more notable in young leaves, suggesting elicitation of a mechanism for grapevine acclimation. By contrast, accumulation of compounds with antioxidant properties, diterpenes α and γ tocopherol and phytol, the sesquiterpene E-nerolidol and the monoterpenes carene, α-pinene and terpinolene had maximum accumulation under high UV-B, which was accentuated in mature leaves. Also the levels of the sesquiterpenic stress-related hormone abscisic acid (ABA) increased under high UV-B, although 24 h post irradiation ABA concentrations decreased. Such increments of antioxidant terpenes along with ABA suggest elicitation of mechanism of defense. The adaptative responses induced by relatively low UV-B irradiations as suggested by synthesis of terpenes related with membrane stability correlated with augments in terpene synthase activity.
Keywords: Grape leaf tissue; Vitis vinifera; Vitaceae; UV-B radiation; Terpene metabolism;

Metabolite profiling reveals novel multi-level cold responses in the diploid model Fragaria vesca (woodland strawberry) by Jens Rohloff; Joachim Kopka; Alexander Erban; Per Winge; Robert C. Wilson; Atle M. Bones; Jahn Davik; Stephen K. Randall; Muath K. Alsheikh (99-109).
Metabolite profiling based on gas chromatography/time-of-flight-mass spectrometry (GC/TOF-MS) was applied to investigate changes in central metabolism in leaves and roots of Fragaria vesca L. (woodland strawberry) under cold acclimation.Display Omitted► Metabolite pools of Fragaria vesca were perturbated upon cold acclimation during a 10-days study. ► Mainly sugars, amino acids and amines were affected. ► Multivariate analyses revealed differences between leaves and roots, genotypes and time points. ► The raffinose pathway was strongly induced upon cold acclimation.Winter freezing damage is a crucial factor in overwintering crops such as the octoploid strawberry (Fragaria × ananassa Duch.) when grown in a perennial cultivation system. Our study aimed at assessing metabolic processes and regulatory mechanisms in the close-related diploid model woodland strawberry (Fragaria vesca  L.) during a 10-days cold acclimation experiment. Based on gas chromatography/time-of-flight-mass spectrometry (GC/TOF-MS) metabolite profiling of three F. vesca genotypes, clear distinctions could be made between leaves and non-photosynthesizing roots, underscoring the evolvement of organ-dependent cold acclimation strategies. Carbohydrate and amino acid metabolism, photosynthetic acclimation, and antioxidant and detoxification systems (ascorbate pathway) were strongly affected. Metabolic changes in F. vesca included the strong modulation of central metabolism, and induction of osmotically-active sugars (fructose, glucose), amino acids (aspartic acid), and amines (putrescine). In contrast, a distinct impact on the amino acid proline, known to be cold-induced in other plant systems, was conspicuously absent. Levels of galactinol and raffinose, key metabolites of the cold-inducible raffinose pathway, were drastically enhanced in both leaves and roots throughout the cold acclimation period of 10 days. Furthermore, initial freezing tests and multifaceted GC/TOF-MS data processing (Venn diagrams, independent component analysis, hierarchical clustering) showed that changes in metabolite pools of cold-acclimated F. vesca were clearly influenced by genotype.
Keywords: Cold acclimation; Compatible solutes; Fragaria × ananassa Duch.; Fragaria vesca  L.; Gas chromatography/time-of-flight-mass spectrometry (GC/TOF-MS); Metabolite profiling; Phenotype;

Mass spectrometry imaging of glucosinolates in Arabidopsis flowers and siliques by Joscelyn Sarsby; Mark W. Towers; Chris Stain; Rainer Cramer; Olga A. Koroleva (110-118).
MALDI-MS imaging in combination with cryo-SEM-EDX analysis demonstrated that glucosinolates are accumulated differentially in specific cells of reproductive organs in Arabidopsis thaliana.Display Omitted► Three glucosinolates were mapped by MALDI-MSI in flower buds, sepals and siliques. ► Two sites of accumulation of glucosinolates were found: S-cells and epidermis of sepals. ► Accumulation of sulphur compounds in S-cells was confirmed by cryo-SEM-EDX.Glucosinolates are multi-functional plant secondary metabolites which play a vital role in plant defence and are, as dietary compounds, important to human health and livestock well-being. Knowledge of the tissue-specific regulation of their biosynthesis and accumulation is essential for plant breeding programs. Here, we report that in Arabidopsis thaliana, glucosinolates are accumulated differentially in specific cells of reproductive organs. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), distribution patterns of three selected compounds, 4-methylsulfinylbutyl (glucoraphanin), indol-3-ylmethyl (glucobrassicin), and 4-benzoyloxybutyl glucosinolates, were mapped in the tissues of whole flower buds, sepals and siliques. The results show that tissue localization patterns of aliphatic glucosinolate glucoraphanin and 4-benzoyloxybutyl glucosinolate were similar, but indole glucosinolate glucobrassicin had different localisation, indicating a possible difference in function. The high resolution images obtained by a complementary approach, cryo-SEM Energy Dispersive X-ray analysis (cryo-SEM-EDX), confirmed increased concentration of sulphur in areas with elevated amounts of glucosinolates, and allowed identifying the cell types implicated in accumulation of glucosinolates. High concentration of sulphur was found in S-cells adjacent to the phloem in pedicels and siliques, indicating the presence of glucosinolates. Moreover, both MALDI MSI and cryo-SEM-EDX analyses indicated accumulation of glucosinolates in cells on the outer surface of the sepals, suggesting that a layer of glucosinolate-accumulating epidermal cells protects the whole of the developing flower, in addition to the S-cells, which protect the phloem. This research demonstrates the high potential of MALDI MSI for understanding the cell-specific compartmentation of plant metabolites and its regulation.
Keywords: Arabidopsis thaliana; Glucosinolates; MALDI MSI; Mass Spectrometry Imaging; Cryo-SEM-EDX; S-cells;

Influence of repeated short-term nitrogen limitations on leaf phenolics metabolism in tomato by Romain Larbat; Kristine M. Olsen; Rune Slimestad; Trond Løvdal; Camille Bénard; Michel Verheul; Frédéric Bourgaud; Christophe Robin; Cathrine Lillo (119-128).
Phenolic compounds contribute to plant defense against pathogens. We show how punctual and repeated nitrogen limitations may enhance phenolic accumulation and regulate phenolic-associated genes expression with limiting effects on plant growth.Display Omitted► Phenolics leaf concentrations rose under short-term nitrogen limitation. ► Phenolic concentration stayed higher than controls after nitrogen resupply. ► Tomato plants do not acclimate to nitrogen limitation. ► Successive nitrogen limitations lead to additive increments in phenolic concentrations.High concentrations of phenolics have been shown to play a role in plant resistance to pathogens. One way to obtain increased phenolic concentrations in plant tissues is to limit mineral nitrogen (N) availability; however, over long periods, this treatment will have a negative effect on plant growth. The aim of our study was to determine the effect of repeated short-term N limitations on plant growth and phenolic metabolism in leaves. Tomato plants (Solanum lycopersicum, cv. Pixie) were subjected to two successive 10-day N-limitation periods (0.15 mM NO3 , 0.01 mM NH4 +), followed by periods of full nutrient supply (15 mM NO3 , 1.2 mM NH4 +). Additionally, other plants were subjected to either of these two limitation periods, and a set of control plants was given a full nutrient supply during the entire period. The phenolic metabolism was monitored by measuring the leaf concentrations of chlorogenic acid, three flavonol glycosides (quercetin and kaempferol derivatives) and two major anthocyanins, together with the expression of eight structural genes and three transcription factors of the phenylpropanoid pathway. The relative growth rate of the plants decreased during the N-limitation periods but was restored as soon as N was resupplied. Each N-limitation period resulted in an up-regulation of the phenolic biosynthetic pathway, as demonstrated by an increase in the leaf phenolic concentration and an up-regulation of the related genes. The genes in the phenolic pathway were down-regulated immediately when N was resupplied; however, the leaf concentrations of several phenolics, particularly flavonol glycosides, were maintained at significantly higher levels than in the control plants for up to 17 days after the end of the first limitation. The amplitude of the increase in leaf phenolic concentration did not depend on the number of N-limitation periods to which the plant was subjected, which indicates that the plants did not acclimate to nitrogen limitation. Successive N-limitation periods resulted in additive increases in flavonol glycoside concentrations.
Keywords: Solanum lycopersicum; Tomato; Nitrogen; Phenolic; Chlorogenic acid; Flavonoids; Anthocyanins; SlMYB12; Rutin; Kaempferol;

Transformation of destruxin B by crucifers and cereals leads to different destruxins, whereas sirodesmin PL is not transformed. Sirodesmin PL is a stronger elicitor of phytoalexins in crucifers.Display Omitted► Destruxin B is transformed by cruciferous species to hydroxydestruxin B. ► Destruxin B is transformed by cereals to four different destruxins. ► Sirodesmin PL is not transformed either by crucifers or cereals. ► Sirodesmin PL is a stronger elicitor of phytoalexins than destruxin B.Destruxin B and sirodesmin PL are phytotoxins produced by the phytopathogenic fungi Alternaria brassicae (Berk.) Sacc. and Leptosphaeria maculans (asexual stage Phoma lingam), respectively. The molecular interaction of destruxin B and sirodesmin PL with cruciferous and cereal species was investigated using HPLC–ESI-MS n . It was determined that crucifers transformed destruxin B to hydroxydestruxin B, but sirodesmin PL was not transformed. Overall, the results suggest that the five cruciferous species Arabidopsis thaliana, Thellungiella salsuginea, Erucastrum gallicum, Brassica rapa and Brassica napus are likely to produce a destruxin B detoxifying enzyme (destruxin B hydroxylase), similar to other cruciferous species reported previously. In addition, HPLC analyses and quantification of the phytoalexins elicited in each cruciferous species by these phytotoxins indicates that sirodesmin PL elicits a larger number of phytoalexins than destruxin B. Interestingly, transformation of destruxin B appears to occur also in the cereals Avena sativa and Triticum aestivum; however, the various destruxin metabolites detected in these cereals suggest that these reactions are non-specific enzymatic transformations, contrary to those observed in crucifers, where only a main transformation pathway is detectable. None of the toxins appear to elicit production of metabolites in either A. sativa or T. aestivum.
Keywords: Alternaria brassicae; Arabidopsis thaliana; Brassicaceae; Cereals; Crucifers; Destruxin B; Leptosphaeria maculans; Phoma lingam; Sirodesmin PL; Thellungiella salsuginea;

Phytotoxic activity in Flourensia campestris and isolation of (−)-hamanasic acid A as its active principle compound by Mariana P. Silva; Leonardo A. Piazza; Daniela López; Marisa J. López Rivilli; Mauricio D. Turco; Juan J. Cantero; Mónica G. Tourn; Ana L. Scopel (140-148).
Phytotoxic activity of leaf aq. extracts from Flourensia campestris (Asteraceae) on lettuce led to isolation of (−)-hamanasic acid A. Its high concentration among the organs and in aq. extracts suggests its putative role as an allelochemical. The essential oils (0.5–1.5 μl ml−1) did not exhibit phytotoxicity against lettuce.Display Omitted► Phytotoxic effects of Flourensia campestris was investigated ► Aq. leaf extracts showed strong inhibition on germination and growth of L.sativa. ► The principal phytocompound isolated was (−)-hamanasic acid A. ► Leaf essential oils did not display phytotoxicity and volatiles were characterized. ► High stores and release of 1 strongly suggest its role as allelochemical.An aqueous extract from Flourensia campestris (Asteraceae) dry aerial parts showed strong inhibition on the germination and growth of Lactuca sativa. Based on bio-guided chromatographic fractionation of aq. extracts from dry and fresh leaves and spectroscopic means, (-)-hamanasic acid A (7-carboxy-8-hydroxy-1(2), 12(13)-dien-bisabolene (1)) was isolated as the most inhibitory active principle on germination (ECg50  = 2.9 mM) and on root (ECr50  = 1.5 mM)/shoot (ECs50  = 2.0 mM) growth. As measured by GC, and correlated with a simple designed 2D-TLC, compound 1 was distributed throughout the plant, with a remarkably high concentration (1.6%) in the leaves and the inflorescences. At least a quarter of the amount of 1 was found in aqueous extracts suggesting that leaching would be a key route for its release into the environment. By contrast, leaf essential oils (HD) between 0.5 and 1.5 μl ml−1 did not show herbicidal effects and 1 was not found in them (TLC) nor among volatiles (HS-SPME). Volatile compositions were assessed by GC-FID and GC–MS and led to the identification of 23 compounds (4 monoterpenes and 19 sesquiterpenes) with a wide seasonal (spring–summer%) variation, represented principally by bicyclo-germacrene (37–6%), spathulenol (4–32%), globulol (20–0%), beta-caryophyllene (15–6%), caryophyllene oxide (1–13%) and bicycloelemene (10–1%), respectively. The high amount of 1 in F. campestris together with its feasibility of being extracted with water suggest that (−)-hamanasic acid A is an allelochemical in this species. Species-specific studies must be carried out to evaluate the potential of 1 as a natural herbicidal compound.
Keywords: Flourensia campestris; Asteraceae; Phytotoxic activity; Allelopathic agent; Sesquiterpenoids; Bisabolanoids; (−)-Hamanasic acid A; Essential oils; Volatiles;

Increased carbon demand brought on by AM fungi affected carbon allocation and partitioning between the different classes of isoprenoids. Carbon resources in AM plants were diverted to carotenoids and chlorophylls which serve base plant functions, instead of volatile isoprenoids like mono- and sesquiterpenes, especially under stress conditions.Display Omitted► We analyze the effects of AM symbiosis on plant isoprenoids production. ► AM symbiosis favoured the production of leaf essential isoprenoids. ► AM fungi root colonization caused a decrease in the root isoprenoid content. ► Drought stress and jasmonic acid application affected plant isoprenoid production. ► Overall, these effects might depend on different partitioning between isoprenoids.Previous studies have shown that root colonization by arbuscular mycorrhiza (AM) fungi enhances plant resistance to abiotic and biotic stressors and finally plant growth. However, little is known about the effect of AM on isoprenoid foliar and root content. In this study we tested whether the AM symbiosis affects carbon resource allocation to different classes of isoprenoids such as the volatile nonessential isoprenoids (monoterpenes and sesquiterpenes) and the non-volatile essential isoprenoids (abscisic acid, chlorophylls and carotenoids). By subjecting the plants to stressors such as drought and to exogenous application of JA, we wanted to test their interaction with AM symbiosis in conditions where isoprenoids usually play a role in resistance to stress and in plant defence. Root colonization by AM fungi favoured the leaf production of essential isoprenoids rather than nonessential ones, especially under drought stress conditions or after JA application. The increased carbon demand brought on by AM fungi might thus influence not only the amount of carbon allocated to isoprenoids, but also the carbon partitioning between the different classes of isoprenoids, thus explaining the not previously shown decrease of root volatile isoprenoids in AM plants. We propose that since AM fungi are a nutrient source for the plant, other carbon sinks normally necessary to increase nutrient uptake can be avoided and therefore the plant can devote more resources to synthesize essential isoprenoids for plant growth.
Keywords: Arbuscular mycorrhiza; Solanum lycopersicum; Jasmonic acid; Isoprenoid; Drought; Abscisic acid; Carotenoids; Monoterpenes; Sesquiterpenes;

Effects of glucosinolates on a generalist and specialist leaf-chewing herbivore and an associated parasitoid by Martine Kos; Benyamin Houshyani; Rafal Wietsma; Patrick Kabouw; Louise E.M. Vet; Joop J.A. van Loon; Marcel Dicke (162-170).
Our study documents a dual defensive role of glucosinolates: glucosinolates conferred resistance against herbivores directly, and indirectly by increasing the performance of their parasitoids.Display Omitted► Effects of glucosinolates on herbivores and parasitoids. ► Negative correlation of glucosinolates with performance of a generalist herbivore. ► Similar result with a specialist herbivore, despite detoxification mechanism. ► Positive correlation with parasitoid performance, perhaps due to host immune system. ► Our study documents a dual defensive role of glucosinolates.Glucosinolates (GLS) are secondary plant metabolites that as a result of tissue damage, for example due to herbivory, are hydrolysed into toxic compounds that negatively affect generalist herbivores. Specialist herbivores have evolved specific adaptations to detoxify GLS or inhibit the formation of toxic hydrolytic products. Although rarely studied, GLS and their breakdown products may also affect parasitoids. The objectives were to test the effects of GLS in a multitrophic system consisting of the generalist herbivore Spodoptera exigua, the specialist herbivore Pieris rapae, and the endoparasitoid Hyposoter ebeninus. Three ecotypes of Arabidopsis thaliana that differ in their GLS composition and concentrations and one transformed line that constitutively produces higher concentrations of aliphatic GLS were used, the latter allowing a direct assessment of the effects of aliphatic GLS on insect performance.Feeding by the generalist S. exigua and the specialist P. rapae induced both higher aliphatic and indole GLS concentrations in the A. thaliana ecotypes, although induction was stronger for indole than aliphatic GLS. For both herbivores a negative correlation between performance and aliphatic GLS concentrations was observed. This suggests that the specialist, despite containing a nitrile-specifier protein (NSP) that diverts GLS degradation from toxic isothiocyanates to less toxic nitriles, cannot completely inhibit the formation of toxic GLS hydrolytic products, or that the costs of this mechanism are higher at higher GLS concentrations. Surprisingly, performance of the parasitoid was positively correlated with higher concentrations of aliphatic GLS in the plant, possibly caused by negative effects on host immune responses. Our study indicates that GLS can not only confer resistance against herbivores directly, but also indirectly by increasing the performance of the parasitoids of these herbivores.
Keywords: Arabidopsis thaliana; Brassicaceae; Hyposoter ebeninus; Multitrophic interactions; Pieris rapae; Spodoptera exigua;

The maize benzoxazinone DIMBOA reacts with glutathione and other thiols to form spirocyclic adducts by David P. Dixon; Jonathan D. Sellars; Alan M. Kenwright; Patrick G. Steel (171-178).
The maize benzoxazinone DIMBOA readily reacts with thiols such as glutathione to form unusual and stable spirocyclic compounds.Display Omitted► Grass benzoxazinones such as DIMBOA readily react with thiols. ► DIMBOA reacts with glutathione to form stable spirocyclic adducts. ► Protein cysteinyl residues similarly react with DIMBOA. ► This reactivity is likely to result in toxicity on ingestion.Maize, wheat and other grasses synthesise large quantities of benzoxazinones and their glucosides, which act as antifeedant and allelopathic agents. These activities are probably due to the electrophilic nature of the aglycones, however, the mechanism of their action is unclear. In biological systems, glutathione (GSH) is the major electrophile-reactive compound so the reaction of the major maize benzoxazinone DIMBOA with GSH was studied. GSH reacts with DIMBOA to form eight isomeric mono-conjugates and eight isomeric di-conjugates. Through NMR studies with the model thiol 2-mercaptoethanol, these were structurally elucidated as unusual spirocycles. Similar reactivity was observed with proteins, with cysteinyl thiols being modified by DIMBOA. The thioether bonds formed were stable and not easily reduced to the parent thiol. DIMBOA can therefore readily deplete GSH levels and irreversibly inactivate enzymes with active-site cysteine residues, with clear implications for potentially toxic effects when young grasses are ingested, whether by insect pests or humans.
Keywords: Zea mays; Triticum aestivum; Electrophile; Thiol reactivity; Benzoxazinone; DIMBOA; Glutathione;

Formation of biphenyl and dibenzofuran phytoalexins in the transition zones of fire blight-infected stems of Malus domestica cv. ‘Holsteiner Cox’ and Pyrus communis cv. ‘Conference’ by Cornelia Chizzali; Mohammed N.A. Khalil; Till Beuerle; Wolfgang Schuehly; Klaus Richter; Henryk Flachowsky; Andreas Peil; Magda-Viola Hanke; Benye Liu; Ludger Beerhues (179-185).
Biphenyl and dibenzofuran phytoalexins were detected in the transition zones of fire blight-infected stems of apple and pear and tested for antibacterial activity against Erwinia amylovora.Display Omitted► Fire blight-infected apple and pear shoots form biphenyls and dibenzofurans. ► The exclusive accumulation site is the transition zone of stems. ► Leaves are devoid of phytoalexins. ► 3,5-Dihydroxybiphenyl exhibits strong antibacterial activity against Erwinia amylovora.In the rosaceous subtribe Pyrinae (formerly subfamily Maloideae), pathogen attack leads to formation of biphenyls and dibenzofurans. Accumulation of these phytoalexins was studied in greenhouse-grown grafted shoots of Malus domestica cv. ‘Holsteiner Cox’ and Pyrus communis cv. ‘Conference’ after inoculation with the fire blight bacterium, Erwinia amylovora. No phytoalexins were found in leaves. However, both classes of defence compounds were detected in the transition zone of stems. The flanking stem segments above and below this zone, which were necrotic and healthy, respectively, were devoid of detectable phytoalexins. The transition zone of apple stems contained the biphenyls 3-hydroxy-5-methoxyaucuparin, aucuparin, noraucuparin and 2′-hydroxyaucuparin and the dibenzofurans eriobofuran and noreriobofuran. In pear, aucuparin, 2′-hydroxyaucuparin, noreriobofuran and in addition 3,4,5-trimethoxybiphenyl were detected. The total phytoalexin content in the transition zone of pear was 25 times lower than that in apple. Leaves and stems of mock-inoculated apple and pear shoots lacked phytoalexins. A number of biphenyls and dibenzofurans were tested for their in vitro antibacterial activity against some Erwinia amylovora strains. The most efficient compound was 3,5-dihydroxybiphenyl (MIC = 115 μg/ml), the immediate product of biphenyl synthase which initiates phytoalexin biosynthesis.
Keywords: Malus domestica; Pyrus communis; Erwinia amylovora; Fire blight; Phytoalexins; Biphenyls; Dibenzofurans; Antibacterial activity;

Genetic and chemical diversity of citron (Citrus medica L.) based on nuclear and cytoplasmic markers and leaf essential oil composition by François Luro; Nicolas Venturini; Gilles Costantino; Julien Paolini; Patrick Ollitrault; Jean Costa (186-196).
The genetic and chemical diversity of the Mediterranean citron was examined with regard to the multiplication and dissemination practices that were related to its uses.Display Omitted► Twenty four citron varieties were studied. ► Study of the polymorphisms of 27 nuclear and cytoplasmic genetic markers. ► Composition of leaf essential oils was determined. ► Relationships were established between varieties, genetics and chemistry.Native to southeast Asia, the citron (Citrus medica L.) was the first citrus fruit to be introduced to the Mediterranean area, in the third century BC, and remained its only citrus representative until the tenth century. The citron was used for its aroma – stemming from its essential oils in leaves and fruit peels – and as symbols in the Jewish religion. Subsequently, the cultivation of citron was extended significantly, peaking in the nineteenth century, when its fruits were used in cosmetics and confectioneries. The objective of this study was to examine the genetic diversity of the Mediterranean citron with regard to the multiplication and dissemination practices that were related to its uses.We studied the polymorphisms of 27 nuclear and cytoplasmic genetic markers of 24 citron varieties, preserved in the citrus germplasm of INRA-CIRAD, San Giuliano, France. The composition of leaf essential oils was determined to establish varieties and phylogenic relationships between accessions. Other major citrus species were included in the molecular analysis, which demonstrated the existence of 13 genetically linked citrons, differing from other citrus species, based on low heterozygosity and specific alleles; these citrons were considered true-type citrons, confirmed by their convergent chemical profiles. We also detected a polymorphism in the chloroplastic genome in these 13 citrons, which, when combined with allelic diversity of 2.4 alleles per locus, suggests that multiple citrons were introduced to the Mediterranean area in last 2 millennia. We determined the genetic origin and relationships of several varieties, such as Corsican, which could have arisen from the selfing of Poncire Commun.We noted a higher-than-expected polymorphism rate among Mediterranean citron varieties, likely due to crossfecundation. The chemical leaf oil composition of several economical varieties, such as Corsican, is distinct and can increase the quality of specific agriculture products for the cosmetics and candy industries.
Keywords: Citrus medica; Citron; cpSSR; Genetic; Essential oil; Leaf; GC; GC–MS;

Proanthocyanidin diversity in the EU ‘HealthyHay’ sainfoin (Onobrychis viciifolia) germplasm collection by Elisabetta Stringano; Christine Hayot Carbonero; Lydia M.J. Smith; Ronald H. Brown; Irene Mueller-Harvey (197-208).
A worldwide Onobrychis viciifolia germplasm collection was screened for proanthocyanidin (PA) content and composition by thiolysis. PA traits clustered into W. European, E. European/Asian and N. American groups and mirrored the morphological and agronomic clusters. The observed, large variation will enable optimising cultivars for nutritional, environmental and veterinary properties.Display Omitted► Proanthocyanidin contents varied 5-fold amongst diverse sainfoin accessions. ► Mean polymer size varied 7-fold. ► Prodelphinidins ranged from 53% to 95% and trans-flavanols from 12% to 34%. ► PA traits generated W. European, E. European/Asian and N. American clusters. ► Clusters based on PA traits resembled those based on morphological traits.This study investigated 37 diverse sainfoin (Onobrychis viciifolia Scop.) accessions from the EU ‘HealthyHay’ germplasm collection for proanthocyanidin (PA) content and composition. Accessions displayed a wide range of differences: PA contents varied from 0.57 to 2.80 g/100 g sainfoin; the mean degree of polymerisation from 12 to 84; the proportion of prodelphinidin tannins from 53% to 95%, and the proportion of trans-flavanol units from 12% to 34%. A positive correlation was found between PA contents (thiolytic versus acid–butanol degradation; P  < 0.001; R 2  = 0.49). A negative correlation existed between PA content (thiolysis) and mDP (P  < 0.05; R 2  = −0.30), which suggested that accessions with high PA contents had smaller PA polymers. Cluster analysis revealed that European accessions clustered into two main groups: Western Europe and Eastern Europe/Asia. In addition, accessions from USA, Canada and Armenia tended to cluster together. Overall, there was broad agreement between tannin clusters and clusters that were based on morphological and agronomic characteristics.
Keywords: Sainfoin; Tannins; Proanthocyanidins; Thiolytic degradation; Germplasm screening;

Iridoid and phenylethanoid glycosides in the New Zealand sun hebes (Veronica; Plantaginaceae) by Rilka M. Taskova; Tetsuo Kokubun; Phil J. Garnock-Jones; Søren R. Jensen (209-217).
From three species of New Zealand Veronica 30 compounds were isolated and identified. Among these were the iridoid glucosides dihydroverminoside and 3,3′,4,4′-tetrahydroxy-α-truxillic acid 6-O-catalpyl diester named heliosepaloside. The chemical markers were used to assess the relationships among sun hebes.Display Omitted► Thirty compounds were isolated and identified from three species of Veronica. ► The iridoids dihydroverminoside and α-truxillic acid catalpyl diester were isolated. ► The species in the group accumulated unusually high levels of verminoside. ► Carbohydrates, iridoids and phenylethanoids were used as chemotaxonomic markers.The sun hebes are a small clade of New Zealand Veronica formerly classified as Heliohebe. The water-soluble compounds of Veronica pentasepala, Veronica raoulii and Veronica hulkeana were studied and 30 compounds including 15 iridoid glucosides, 12 phenylethanoid glycosides, the acetophenone glucoside pungenin, the mannitol ester hebitol II and mannitol were isolated. Of these, five were previously unknown in the literature: dihydroverminoside and 3,3′,4,4′-tetrahydroxy-α-truxillic acid 6-O-catalpyl diester, named heliosepaloside, as well as three phenylethanoid glycoside esters heliosides D, E and F, all derivatives of aragoside. The esters of cinnamic acid derivatives with iridoid and phenylethanoid glycosides and an unusually high concentration of verminoside were found to be the most distinctive chemotaxonomic characters of the sun hebes. The chemical profiles of the species were compared and used to assess the phylogenetic relationships in the group.
Keywords: Veronica; Heliohebe; Iridoid glucosides; Phenylethanoid glycosides; Chemotaxonomy; Dihydroverminoside; Heliosepaloside; α-Truxillic ester; Heliosides D, E, F;

Phloroglucinol derivatives from Hypericum empetrifolium with antiproliferative activity on endothelial cells by Sebastian Schmidt; Guido Jürgenliemk; Helen Skaltsa; Jörg Heilmann (218-225).
Five acylphloroglucinols substituted with monoterpenoids were isolated from a petrol ether extract of Hypericum empetrifolium by an 1H NMR guided approach. Inhibition of cell proliferation was measured using a human microvascular endothelial cell line. Most active compounds showed a concentration dependent inhibition of cell proliferation with IC50 values in the lower micromolar range.Display Omitted► We isolated five phloroglucinol derivatives from Hypericum empetrifolium. ► We applied a 1H NMR guided approach. ► We found a rare monoterpenoid substitution of the phloroglucinols. ► We found significant inhibition of cell proliferation in endothelial cells.Five acylphloroglucinols substituted with monoterpenoids (empetrifelixin A–D and empetrikajaforin), three known monocyclic acylphloroglucinols and one monocyclic acylphloroglucinol were isolated from a petrol ether extract of Hypericum empetrifolium after fractionation by flash chromatography on silica gel, RP-18 and subsequent purification by preparative HPLC (RP-18). Their structures were elucidated by 1D, 2D NMR techniques and HREIMS. To determine a possible anti-angiogenic activity, inhibition of cell proliferation was measured using a human microvascular endothelial cell line (HMEC-1). Subconfluent grown HMEC-1 cells were treated with all compounds isolated in sufficient amounts and stained with crystal violet. Highest activity was observed for empetrifelixin A and empetrifelixin D showing a concentration dependent inhibition of cell proliferation with IC50 values of 6.5 ± 0.1 and 7.3 ± 0.4 μM, respectively. Empetrifelixin A also showed activity in a cell migration assay with HMEC-1 cells in low micromolar concentrations.
Keywords: Hypericum empetrifolium; Hypericaceae; Acylphloroglucinols; Anti-angiogenic activity; NMR guided fractionation;

Isolation and characterization of ellagitannins as the major polyphenolic components of Longan (Dimocarpus longan Lour) seeds by Yuttana Sudjaroen; William E. Hull; Gerhard Erben; Gerd Würtele; Supranee Changbumrung; Cornelia M. Ulrich; Robert W. Owen (226-237).
The major polyphenolic antioxidant components of Longan seeds are identified as corilagin, chebulagic acid, isomallotinic acid and geraniin.Display Omitted► This is a comprehensive report of phenolic compounds present in Longan seeds. ► Phenolic compounds were identified by HPLC–ESI-MS, nano-ESI-MS–MS and NMR (600 MHz). ► Complete and unequivocal NMR data is presented for the purified major components. ► The extracts and purified phenolic compounds displayed strong antioxidant activity.Longan (Dimocarpus longan Lour, syn. Euphoria longan Lam.) represents an important fruit in Northern Thailand and has significant economic impact. The fruit is either consumed fresh or as commercially prepared dried and canned products. The canning industry in Thailand produces considerable quantities of waste products, in particular Longan seeds. Because these seeds may be an exploitable source of natural phenolic antioxidants, it was of interest to identify, purify and quantitate the major potential antioxidant phenolics contained therein. The polyphenolic fraction from ground Longan seeds was obtained by extraction with methanol after delipidation with hexane. The hexane extract contained predominantly long-chain fatty acids with major contributions from palmitic (35%) and oleic (28%) acids. The polyphenolic fraction (80.90 g/kg dry weight) was dominated by ellagic acid (25.84 g/kg) and the known ellagitannins corilagin (13.31 g/kg), chebulagic acid (13.06 g/kg), ellagic acid 4-O-α-l-arabinofuranoside (9.93 g/kg), isomallotinic acid (8.56 g/kg) and geraniin (5.79 g/kg). Structure elucidation was performed with mass spectrometry and complete assignment of 1H and 13C NMR signals. The methanol extracts exhibited strong antioxidant capacities with an IC50 of 154 μg/ml for reactive oxygen species attack on salicylic acid and 78 μg/ml for inhibition of xanthine oxidase in the hypoxanthine/xanthine oxidase assay. The extracts were less effective in the 2-deoxyguanosine assay (IC50  = 2.46 mg/ml), indicating that gallates along with ellagic acid and its congeners exert their potential antioxidant effects predominantly by precipitation of proteins such as xanthine oxidase. This was confirmed for the pure compounds gallic acid, methyl gallate, ellagic acid and corilagin.
Keywords: Dimokarpus; Sapindaceae; Longan; Chebulagic acid; Corilagin; Ellagic acid; Ellagic acid 4-O-α-l-arabinofuranoside; Geraniin; HPLC–ESI-MS; Isomallotinic acid; mass spectrometry; NMR;

Flavonoid and cardenolide glycosides and a pentacyclic triterpene from the leaves of Nerium oleander and evaluation of cytotoxicity by Bina Shaheen Siddiqui; Nasima Khatoon; Sabira Begum; Ahsana Dar Farooq; Kehkashan Qamar; Huma Aslam Bhatti; Syed Kashif Ali (238-244).
A triterpene (1), two flavonoidal glycosides (2 and 3), a cardenolide (4), and 11 known compounds, were isolated from Nerium oleander leaves (Apocynaceae). Eight compounds were evaluated against the MCF-7 human breast cancer cell line. Three compounds were further assayed using a panel of 57 human cancer cell lines.Display Omitted► Isolation of flavonoids, triterpenes and cardenolides from Nerium oleander leaves. ► Structure elucidation by TOFESIMS and 1D and 2D NMR spectroscopy. ► Cytotoxicity assays of adynerin, oleandrin, odoroside A and B against MCF-7 cells. ► Evaluation of oleandrin, odoroside A and B against 57 cell lines of human cancer. ► First report of cytotoxicity of oleandrin, odoroside A and B against 57 cell lines.A pentacyclic triterpene, oleanderocioic acid, two flavonoidal glycosides, quercetin-5-O-[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside and kaempferol-5-O-[α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside, and a cardenolide, oleandigoside, together with 11 known compounds, were isolated from the leaves of Nerium oleander. Their structures were elucidated on the basis of spectroscopic analysis. The growth inhibitory and cytotoxic activities of eight compounds were evaluated against the MCF-7 human breast cancer cell line using a sulforhodamine B assay. Three compounds, oleandrin, odoroside A and B were further assayed using a panel of 57 human cancer cell lines.
Keywords: Nerium oleander; Apocynaceae; Pentacyclic triterpene; Flavonoidal glycosides; Cardenolides; Structure and cytotoxicity;

Afritoxinones A and B, dihydrofuropyran-2-ones produced by Diplodia africana the causal agent of branch dieback on Juniperus phoenicea by Antonio Evidente; Marco Masi; Benedetto T. Linaldeddu; Antonio Franceschini; Bruno Scanu; Alessio Cimmino; Anna Andolfi; Andrea Motta; Lucia Maddau (245-250).
Afritoxinones A and B, two phytotoxic dihydrofuropyran-2-ones, were isolated together with oxysporone from Diplodia africana, a fungal pathogen responsible for branch dieback of Juniperus phoenicea.Display Omitted► Afritoxinones A and B, bioactive furopyran-2-ones, were isolated from Diplodia africana. ► Plant-pathogen interaction have importance for environmental and forestall heritage. ► Phytotoxins are useful in taxonomic classification of phytopathogenic fungi.Two phytotoxic dihydrofuropyran-2-ones, named afritoxinones A and B, were isolated from liquid culture of Diplodia africana, a fungal pathogen responsible for branch dieback of Phoenicean juniper in Italy. Additionally, six others known metabolites were isolated and characterized: oxysporone, sphaeropsidin A, epi-sphaeropsidone, R-(−)-mellein, (3R,4R)-4-hydroxymellein and (3R,4S)-4-hydroxymellein. The structures of afritoxinones A and B were established by spectroscopic and optical methods and determined to be as (3aS ,6R ,7aS)-6-methoxy-3a,7a-dihydro-3H,6H-furo[2,3-b]pyran-2-one and (3aR ,6R ,7aS)-6-methoxy-3a,7a-dihydro-3H,6H-furo[2,3-b]pyran-2-one, respectively. The phytotoxic activity of afritoxinones A and B and oxysporone was evaluated on host (Phoenicean juniper) and non-host plant (holm oak, cork oak and tomato) by cutting and leaf puncture assay. Oxysporone proved to be the most phytotoxic compound. This study represents the first report of secondary metabolites produced by D. africana. In addition, the taxonomic implications of secondary metabolites in Botryosphaeriaceae family studies are discussed.
Keywords: Juniperus phoenicea; Botryosphaeriaceae; Diplodia africana; Phytotoxins; Afritoxinones A and B;

Prenylated cinnamate and stilbenes from Kangaroo Island propolis and their antioxidant activity by Abdallah Abu-Mellal; Nooshin Koolaji; Rujee K. Duke; Van H. Tran; Colin C. Duke (251-259).
One novel prenylated cinnamic acid derivative and six novel prenylated stilbenes were isolated from the ethyl acetate extract of Kangaroo Island propolis from Australia. The unique sample consisted of approximately 75% (excluding wax) of prenylated stilbenes most of them related to the tetrahydroxystilbene; piceatannol.Display Omitted► A novel cinnamic acid derivative and six novel prenylated stilbenes were isolated from Kangaroo Island propolis. ► Seventy five percent of the total phenolics of Kangaroo Island propolis were prenylated stilbenes. ► High level of O- and C-prenylation were present in Kangaroo Island propolis. ► Kangaroo Island propolis showed a stronger DPPH free radical scavenging activity than Brazilian green propolis.A prenylated cinnamic acid derivative as well as six prenylated tetrahydroxystilbenes were isolated from the ethyl acetate extract of propolis that originated from Kangaroo Island, Australia. Furthermore, six known stilbenes and two known flavanones were also identified from the same sample. Stilbenes are not common in propolis; therefore, Kangaroo Island propolis is considered a unique type of propolis that is rich in prenylated stilbenes. Stilbene propolis from Kangaroo Island showed a stronger scavenging activity towards DPPH free radical than Brazilian green propolis. This strong activity can be explained by the presence of large number of stilbenes, most of them showed strong free radical scavenging activity.
Keywords: Propolis; Honeybee; Cinnamates; Stilbenes; Prenylated compounds; Piceatannol; Antioxidant activity;

Biosynthesis of rhodiocyanosides in Lotus japonicus: Rhodiocyanoside A is synthesized from (Z)-2-methylbutanaloxime via 2-methyl-2-butenenitrile by Shigeki Saito; Mohammed Saddik Motawia; Carl Erik Olsen; Birger Lindberg Møller; Søren Bak (260-267).
Biosynthesis of rhodiocyanosides in Lotus japonicus. 2-Methyl-2-butenenitrile is a new intermediate derived from (Z)-2-methylbutanaloxime (Ile-ox) by a cytochrome(s) P450 monooxygenase.Display Omitted► 2-Methyl-2-butenenitrile is new intermediate in biosynthesis of rhodiocyanosides. ► The aglycon of rhodiocyanoside A cyclizes to 3-methyl-2(5H)-furanone. ► Biosynthesis of cyanogenic glucosides and rhodiocyanosides branch at Ile-oxime. ► Transformation of Ile-ox to 2-methyl-2-butenenitrile is catalyzed by a P450.Lotus japonicus contains the two cyanogenic glucosides, linamarin and lotaustralin, and the non cyanogenic hydroxynitriles, rhodiocyanoside A and D, with rhodiocyanoside A as the major rhodiocyanoside. Rhodiocyanosides are structurally related to cyanogenic glucosides but are not cyanogenic. In vitro administration of intermediates of the lotaustralin pathway to microsomes prepared from selected L. japonicus accessions identified 2-methyl-2-butenenitrile as an intermediate in the rhodiocyanoside biosynthetic pathway. In vitro inhibitory studies with carbon monoxide and tetcyclacis indicate that the conversion of (Z)-2-methylbutanal oxime to 2-methyl-2-butenenitrile is catalyzed by cytochrome P450(s). Carbon monoxide inhibited cyanogenic glucosides as well as rhodiocyanosides synthesis, but inhibition of the latter pathway was much stronger. These results demonstrate that the cyanogenic glucoside and rhodiocyanosides pathways share CYP79Ds to obtain (Z)-2-methylbutanaloxime from l-isoleucine, whereas the subsequent conversions are catalyzed by different P450s. The aglycon of rhodiocyanoside A forms the cyclic product 3-methyl-2(5H)-furanone. Furanones are known to possess antimicrobial properties indicating that rhodiocyanoside A may have evolved to serve as a phytoanticipin that following β-glucosidase activation and cyclization of the aglycone formed, give rise to a potent defense compound.
Keywords: Lotaustralin; Hydroxynitrile glucosides; Cyanogenic glucosides; Rhodiocyanosides; Bioactive compounds; Plant defense; 2-Methyl-2-butenenitrile; 3-Methyl-2(5H)-furanone;

Triterpene saponins from Antonia ovata leaves by Abdulmagid Alabdul Magid; Nathalie Lalun; Christophe Long; Nicolas Borie; Hélène Bobichon; Christian Moretti; Catherine Lavaud (268-274).
Six saponins were isolated from the leaves of Antonia ovata. Their structures were determined by physical and NMR analysis and their cytotoxicity against KB cell line were evaluated.Display Omitted► Six saponins from Antonia ovata. ► All include a pentasaccharide linked at C-3 of polyhydroxyoleanene triterpenoids. ► Cytotoxicity were evaluated against KB cell line. ► The role of ester part in cytotoxicity was discussed.Six pentacyclic triterpenoid saponins, named antoniosides E–J along with two known alkaloids, were isolated from the leaves of Antonia ovata. Their structures were determined by the extensive use of 1D and 2D-NMR experiments along with HRESIMS analysis and acid hydrolysis. All isolated saponins contained the same pentasaccharide chain: 3-O-[β-d-glucopyranosyl-(1 → 2)]-[β-d-glucopyranosyl-(1 → 4)]-[β-d-glucopyranosyl-(1 → 3)-α-l-arabinopyranosyl(1 → 6)]-β-d-glucopyranoside, linked at C-3 of esterified derivatives of polyhydroxyoleanene triterpenoids (theasapogenol A and 15α-hydroxy-theasapogenol A). Isolated compounds were evaluated for their cytotoxic activity against KB cell line by a WST-1 assay, and the IC50 values ranged from 3.3 to 5.3 μM.
Keywords: Antonia ovata; Loganiaceae; Triterpenoid saponins; Theasapogenol A; 15α-Hydroxy-theasapogenol A; Cytotoxic activity;

Rare noriridoids from the roots of Andrographis paniculata by Chong Xu; Gui-Xin Chou; Chang-Hong Wang; Zheng-Tao Wang (275-279).
Andrographidoid A–E (15) rare noriridoids were isolated from roots of Andrographis paniculata. The noriridoids 14 have (semi-) acetal structures at C-3 but not at C-1.Display Omitted► Five rare iridoids (nor-monoterpenes) were isolated from genus Andrographis. ► Compounds 14 had (semi) acetal structures located at C-3 but not at C-1.The rare noriridoids, Andrographidoids A–E (15), along with a known iridoid curvifloruside F (6), were isolated from roots of Andrographis paniculata. All noriridoids were aglycones and 14 had (semi-) acetal structures located at C-3 but not at C-1. Their structures were established by a series of 1D and 2D NMR analyses. The antibacterial activity of these iridoids was also assessed using the microtitre plate broth dilution method.
Keywords: Andrographis paniculata; Acanthaceae; Iridoid; Noriridoids; Andrographidoids A–E;

Xanthones from the stem bark of Garcinia bracteata with growth inhibitory effects against HL-60 cells by Sheng-Li Niu; Zhan-Lin Li; Feng Ji; Gu-Yue Liu; Nan Zhao; Xiao-Qiu Liu; Yong-Kui Jing; Hui-Ming Hua (280-286).
Five new xanthones together with twenty-six known xanthones were isolated from the stem bark of Garcinia bracteata. The growth inhibitory activities of these compounds were evaluated against human leukaemic HL-60 cell line.Display Omitted► Constituents of stem bark of Garcinia bracteata were investigated. ► Thirty one xanthones were isolated from the EtOH extract, five hitherto unknown. ► A hypothetical biosynthetic pathway linking 14 was presumed. ► The growth inhibitory activities of the isolated compounds were measured in vitro. ► A preliminary structure–activity relationship was discussed.Five xanthones, 1,4,5,6-tetrahydroxyxanthone (1) and bracteaxanthones III–VI (25) together with twenty-six known compounds (631), were isolated from the ethanol extract of the stem bark of Garcinia bracteata. Their structures were elucidated via spectroscopic analyses. Growth inhibitory activities of these compounds against the human leukaemic HL-60 cell line were measured in vitro. Compounds 7, 11, and 29 exhibited moderate activities with GI50 values of 2.8, 3.4, and 3.1 μM, respectively, and a preliminary structure–activity relationship is discussed.
Keywords: Garcinia bracteata; Clusiaceae; Xanthone; Cell growth inhibition; HL-60 cell line; Bracteaxanthone;

Identification of intermediates involved in the biosynthetic pathway of 3-mercaptohexan-1-ol conjugates in yellow passion fruit (Passiflora edulis f. flavicarpa) by Bruno Fedrizzi; Graziano Guella; Daniele Perenzoni; Mattia Gasperotti; Domenico Masuero; Urska Vrhovsek; Fulvio Mattivi (287-293).
The identification of the missing steps in S-glutathionyl-3-mercaptohexan-1-ol degradation pathways leads to better understanding of the formation of the tropical aroma of yellow passion fruit.Display Omitted► New synthetic strategy for the preparation of Glu-Cys-3MH and Cys-Gly-3MH. ► The three missing stages leading to the S-cysteinylated precursor were identified. ► Two intermediates found between S-glutathionyl and S-cysteinyl 3-mercaptohexan-1-ol. ► Demonstrating that the plant is capable of activating both metabolic routes.Yellow passion fruit is one of the most well-known tropical fruits and much of its success comes from its typical aroma. Key compounds in explaining yellow passion fruit scent are volatile thiols. These molecules are reported to be present in several fruits and originate from non-volatile precursors. Such free thiols are particularly appreciated in white wines and considerable efforts have been made to try to maximise their production and understand their biosynthesis.Two main precursors have been identified so far: S-glutathionylated and S-cysteinylated precursors, the latter originating in the breaking down of the glycyl and glutamyl moieties of the former. Improving knowledge about this pathway is currently one of the main challenges in the field of aroma chemistry.Only S-cysteinylated precursors have been reported in the literature for yellow passion fruit, thus much of the biochemical pathway remains unknown.In this paper a combination of organic synthesis, MS and NMR experiments was developed in order to investigate this pathway in yellow passion fruit. The three missing stages leading to the S-cysteinylated precursor were clearly identified. Both intermediate species between S-glutathionyl and S-cysteinyl 3-mercaptohexan-1-ol were found, suggesting that the plant is capable of activating both metabolic routes.The information gained would appear to be crucial for study of this important pathway and for potentially extending this knowledge to other plants, in particular the grapevine.
Keywords: Yellow passion fruit; Thiols; Glutathionyl-precursor; Cysteinyl-precursor; MS; NMR;

Phytochemical investigation of Vitis wilsonae Veitch resulted in isolation of three oligostilbenes (13) and a biomimetic oligostilbene 4, whose structures were identified by 1D, 2D NMR spectroscopic and chemical means. All compounds showed selective quenching capacities towards 1O2.Display Omitted► HPLC/ESI-MSn-guided isolation has been utilized to obtain compounds of different molecular masses. ► Four compounds including a biomimetic one were obtained from Vitis wilsonae. ► A biogenetic transformation has been achieved by HRP–H2O2, acid, and UV irradiation treatment. ► The compounds showed selective quenching effects towards 1O2 compared with less effect on O 2 · − and OH. ► The compounds showed potent inhibitory effects in response to oxidative stress caused by 1O2.Two previously unknown resveratrol trimers named wilsonols A–B, as well as a resveratrol tetramer named wilsonol C, were isolated from Vitis wilsonae Veitch, together with 12 known oligostilbenes. Their chemical structures have been elucidated by detailed analyses of 1D and 2D NMR spectroscopic data, as well as chemical evidence obtained via either catalysis with HRP (horseradish peroxidase) and H2O2 (hydrogen peroxide), acid, or UV irradiation. During the chemical processes, a biomimetic resveratrol tetramer named diviniferin B that has not been found in nature was obtained. These oligostilbenes showed potent scavenging abilities towards DPPH radicals and selective quenching effects on 1O2 radicals. Furthermore, the biogenetic transformations between the 16 oligostilbenes have been elaborated chemically to provide a comprehensive mechanism of the antioxidative defense system in this plant species.
Keywords: Vitis wilsonae Veitch; Vitaceae; Oligostilbene; Wilsonols A–C; Diviniferin B; Biogenetic transformation; DPPH; 1O2 (singlet oxygen); Antioxidative defense system;

α-Santalane type sesquiterpenes were isolated from the heartwood of Santalum album. Sesquiterpenoid 1 was found to show tumor selective cytotoxic activity and induce caspase-dependent apoptotic cell death in HL-60 cells.Display Omitted► α-Santalol derivatives were isolated from Santalum album heartwood. ► Fourteen compounds were evaluated for their cytotoxicity against HL-60 and TIG-3cells. ► (9S,10E)-9-hydroxy-α-santalal showed tumor selective cytotoxicity. ► It also induced caspase-dependent apoptotic cell death in HL-60 cells.A bioassay-guided fractionation of the heartwood of Santalum album led to the isolation of seven α-santalol derivatives including (9S,10E)-9-hydroxy-α-santalal, (10R,11S)-10,11-dihydroxy-α-santalol, (9E)-11,13-dihydroxy-α-santalol, and (10E)-12-hydroxy-α-santalic acid. Their structures were determined on the basis of results of spectroscopic analysis, including two-dimensional (2D) NMR spectroscopic data. The isolated compounds and derivatives were evaluated for cytotoxicity against HL-60 human promyelocytic leukemia cells and TIG-3 normal human diploid fibroblasts. Of these (9S,10E)-9-hydroxy-α-santalal, exhibited tumor-selective cytotoxicity. The apoptosis induction properties of sesquiterpenes with cytotoxic potency in HL-60 cells are also described.
Keywords: Santalum album; Santalaceae; Sesquiterpene; α-Santalol; HL-60 cells; Cytotoxicity;

Chlorantholides A–F, eudesmane-type sesquiterpene lactones from Chloranthus elatior by Fei Wang; Dong-Sheng Zhou; Guo-Zhu Wei; Fu-Cai Ren; Ji-Kai Liu (312-317).
Six eudesmane sesquiterpene lactones, chlorantholides A–F (16), were isolated from Chloranthus elatior. The structure of an eudesmanolide from C. anhuiensis: 8β-hydroxy-1-oxoeudesma-3,7(11)-dien-12,8-olide (4a), was revised as 8β-hydroxy-2-oxoeudesma-3,7(11)-dien-12,8-olide (chlorantholide D, 4).Display Omitted► Six eudesmane sesquiterpene lactones were isolated from Chloranthus elatior. ► The structure of an eudesmanolide was revised. ► The HMBC and ROESY correlations of hydroxy proton signals were applied for structure elucidation. ► The CD exciton chirality method was employed to determine absolute configuration.Six eudesmane-type sesquiterpene lactones, named chlorantholides A–F, were isolated from the ethanol extract of Chloranthus elatior (Chloranthaceae) together with 12 known compounds. Their structures were elucidated on the basis of extensive spectroscopic analysis, and their absolute configurations were studied by the CD exciton chirality method. The structure of a recently reported eudesmanolide from Chloranthus anhuiensis: 8β-hydroxy-1-oxoeudesma-3,7(11)-dien-12,8-olide, was also revised as 8β-hydroxy-2-oxoeudesma-3,7(11)-dien-12,8-olide (chlorantholide D).
Keywords: Chloranthaceae; Chloranthus elatior; Eudesmanolide; Sesquiterpene; CD exciton chirality method; Structure revision;