Phytochemistry (v.117, #C)

Structure and cytotoxic activity of sesquiterpene glycoside esters from Calendula officinalis L.: Studies on the conformation of viridiflorol by Michele D’Ambrosio; Alexandru Ciocarlan; Elisa Colombo; Antonio Guerriero; Cosimo Pizza; Enrico Sangiovanni; Mario Dell’Agli (1-9).
The sesquiterpene glycoside esters are responsible for cytotoxic effects of extracts of C. officinalis. Their structures were established by spectroscopic methods, chemical reactions and molecular mechanic calculations.Display OmittedTopic applications of Calendula officinalis L. lipophilic extracts are used in phytotherapy to relieve skin inflammatory conditions whereas infusions are used as a remedy for gastric complaints. Such a different usage might be explained by some cytotoxicity of lipophilic extracts at gastric level but little is known about this. Therefore, we screened the CH2Cl2 extract from the flowers of C. officinalis by MTT and LDH assays in human epithelial gastric cells AGS. This bioassay-oriented approach led to the isolation of several sesquiterpene glycosides which were structurally characterized by spectroscopic measurements, chemical reactions and MM calculations. The conformational preferences of viridiflorol fucoside were established and a previously assigned stereochemistry was revised. The compounds 1a, 2a and 3f showed comparably high cytotoxicity in the MTT assays, whereas the effect on LDH release was lower. Our study provides new insights on the composition of C. officinalis extracts of medium polarity and identifies the main compounds that could be responsible for cytotoxic effects at gastric level.
Keywords: Calendula officinalis; Marigold; Sesquiterpene glycoside; Viridiflorol; Ledol; α-Elemol; β-Eudesmol; Officinoside D;

Entonalactams A–C: Isoindolinone derivatives from an Australian rainforest fungus belonging to the genus Entonaema by Vanida Choomuenwai; Karren D. Beattie; Peter C. Healy; Katherine T. Andrews; Nigel Fechner; Rohan A. Davis (10-16).
Bioassay-guided fractionation of an anti-malarial extract derived from the Australian rainforest fungus Entonaema sp. resulted in the isolation and characterisation of three new isoindolinone derivatives, entonalactams A–C.Display OmittedBioassay-guided fractionation of an antimalarial DCM/MeOH extract derived from the Australian rainforest fungus Entonaema sp. resulted in the isolation of three new isoindolinone derivatives, entonalactams A–C (13), along with the known natural products 3-methoxy-5-methylbenzene-1,2-diol (4), daldinal B (5), and ergosta-4,6,8(14),22-tetraen-3-one (6). The chemical structures of the new secondary metabolites were determined following extensive 1D/2D NMR and MS data analysis. A single crystal X-ray structure for entonalactam A (1) confirmed the NMR-based structure assignment. Entonalactams A–C (1–3) were all determined to be racemic based on chiro-optical data. All secondary metabolites were tested in vitro against Plasmodium falciparum malaria parasites, and ergosta-4,6,8(14),22-tetraen-3-one (6) was identified as the most active compound with 66% inhibition at 50 μM.
Keywords: Entonaema; Isoindolinone; Lactam; Entonalactam; Rainforest; Fungi; Natural product; Malaria;

Cell-specific analysis of Heliotropium indicum was used to identify candidate genes of pyrrolizidine alkaloid biosynthesis.Display OmittedProgress has recently been made in the elucidation of pathways of secondary metabolism. However, because of its diversity, genetic information concerning biosynthetic details is still missing for many natural products. This is also the case for the biosynthesis of pyrrolizidine alkaloids. To close this gap, we tested strategies using tissues that express this pathway in comparison to tissues in which this pathway is not expressed. As many pathways of secondary metabolism are known to be induced by jasmonates, the pyrrolizidine alkaloid-producing species Heliotropium indicum, Symphytum officinale, and Cynoglossum officinale of the Boraginales order were treated with methyl jasmonate. An effect on pyrrolizidine alkaloid levels and on transcript levels of homospermidine synthase, the first specific enzyme of pyrrolizidine alkaloid biosynthesis, was not detectable. Therefore, a method was developed by making use of the often observed cell-specific production of secondary compounds. H. indicum produces pyrrolizidine alkaloids exclusively in the shoot. Homospermidine synthase is expressed only in the cells of the lower leaf epidermis and the epidermis of the stem. Suggesting that the whole pathway of pyrrolizidine alkaloid biosynthesis might be localized in these cells, we have isolated single cells of the upper and lower epidermis by laser-capture microdissection. The resulting cDNA preparations have been used in a subtractive transcriptomic approach. Quantitative real-time polymerase chain reaction has shown that the resulting library is significantly enriched for homospermidine-synthase-coding transcripts providing a valuable source for the identification of further genes involved in pyrrolizidine alkaloid biosynthesis.
Keywords: Evolution; Homospermidine synthase; Laser-capture microdissection; Secondary metabolism; Specialized metabolism; Suppression subtractive hybridization;

An IAA-glucosyltransferase (IAGlc synthase) catalyzing the conjugation of indole-3-acetic acid (IAA) with glucose in seeds of Pisum sativum was isolated and purified. The IAGlc synthase is involved in modification of glycoproteins from pea seeds.Display OmittedThe glycosylation of auxin is one of mechanisms contributing to hormonal homeostasis. The enzyme UDPG: indole-3-ylacetyl-β-d-glucosyltransferase (IAA glucosyltransferase, IAGlc synthase) catalyzes the reversible reaction: IAA + UDPG ↔ 1-O-IA-glucose + UDP, which is the first step in the biosynthesis of IAA-ester conjugates in monocotyledonous plants. In this study, we report IAA-glucosyltransferase isolated using a biochemical approach from immature seed of pea (Pisum sativum). The enzyme was purified by PEG fractionation, DEAE-Sephacel anion-exchange chromatography and preparative PAGE. LC–MS/MS analysis of tryptic peptides of the enzyme revealed the high identity with maize IAGlc synthase, but lack of homology with other IAA-glucosyltransferases from dicots. Biochemical characterization showed that of several acyl acceptors tested, the enzyme had the highest activity on IAA as the glucosyl acceptor (K m  = 0.52 mM, V max  = 161 nmol min−1, k cat/K m  = 4.36 mM s−1) and lower activity on indole-3-propionic acid and 1-naphthalene acetic acid. Whereas indole-3-butyric acid and indole-3-propionic acid were competitive inhibitors of IAGlc synthase, d-gluconic acid lactone, an inhibitor of β-glucosidase activity, potentiated the enzyme activity at the optimal concentration of 0.3 mM. Moreover, we demonstrated that the 1-O-IA-glucose synthesized by IAGlc synthase is the substrate for IAA labeling of glycoproteins from pea seeds indicating a possible role of this enzyme in the covalent modification of a class of proteins by a plant hormone.
Keywords: Auxin conjugate; Indole-3-acetic acid; Indole-3-ylacetyl-β-d-glucose; Glucosylation; Glucosyltransferase; Pisum sativum;

Diacylglyceryltrimethylhomoserine content and gene expression changes triggered by phosphate deprivation in the mycelium of the basidiomycete Flammulina velutipes by Svetlana V. Senik; Liliya G. Maloshenok; Ekaterina R. Kotlova; Alexey L. Shavarda; Konstantin V. Moiseenko; Sergey A. Bruskin; Olga V. Koroleva; Nadezhda V. Psurtseva (34-42).
We present our results of a study examining membrane lipids and expression analysis of the gene for betaine lipid synthase (BTA1) and genes for PC biosynthesis (CHO2 and CPT1) during phosphate starvation of Flammulina velutipes. The phylogenetic relationships between putative orthologs of BTA1 proteins from other basidiomycete fungi are discussed.Display OmittedDiacylglyceryltrimethylhomoserines (DGTS) are betaine-type lipids that are phosphate-free analogs of phosphatidylcholines (PC). DGTS are abundant in some bacteria, algae, primitive vascular plants and fungi. In this study, we report inorganic phosphate (Pi) deficiency-induced DGTS synthesis in the basidial fungus Flammulina velutipes (Curt.: Fr.) Sing. We present results of an expression analysis of the BTA1 gene that codes for betaine lipid synthase and two genes of PC biosynthesis (CHO2 and CPT1) during phosphate starvation of F. velutipes culture. We demonstrate that FvBTA1 gene has increased transcript abundance under phosphate starvation. Despite depletion in PC, both CHO2 and CPT1 were determined to have increased expression. We also describe the deduced amino acid sequence and genomic structure of the BTA1 gene in F. velutipes. Phylogenetic relationships between putative orthologs of BTA1 proteins of basidiomycete fungi are discussed.
Keywords: Flammulina velutipes; Basidiomycetes; Betaine lipid; Glycerolipid metabolism; Phosphate starvation; Gene expression; BTA1; CHO2; CPT1; Phylogenetic analysis;

Methyl jasmonate, salicylic acid, glucose and wounding, factors mimicking biotic stress, enhances the accumulation of defense compound glucosinolates in Brassica juncea through co-ordinated interplay of glucosinolate regulators.Display OmittedGlucosinolates are nitrogen and sulfur containing secondary metabolites found mainly in the Brassicaceae. They function as plant defense compounds against a broad spectrum of pathogens and pests. Since these molecules form part of the plant defense mechanism, glucosinolate biosynthesis may be modulated by environmental signals leading to activation of a biological stress response. In the current study, we have mimicked such conditions by exogenously applying biotic elicitors such as methyl jasmonate, salicylic acid, glucose and mechanical injury in Brassica juncea seedling over a time course experiment. We found that total glucosinolates over-accumulated under these stress conditions with maximum accumulation observed 24 h post treatment. Indole glucosinolates like 1-methoxy-indol-3-ylmethyl and its precursor indol-3-methyl glucosinolates showed a more significant induction compared to aliphatic glucosinolates thereby suggesting a prominent role of indole glucosinolates during plant defense response in B. juncea seedlings. In contrast, the higher amounts of aliphatic glucosinolates were less regulated by the tested biotic elicitors in B. juncea. Expression profiling of multiple homologs of key transcriptional regulators of glucosinolate biosynthesis further showed that a complex interplay of these regulators exists in polyploid B. juncea where they exert co-ordinated and overlapping effects toward altering glucosinolate accumulation. This study has a significant role toward understanding and augmenting plant defense mechanisms in B. juncea, a globally important oilseed crop of genus Brassica.
Keywords: Biotic elicitors; Brassica juncea; Glucose; Glucosinolates; MYB genes; Plant defense; Wounding;

Toxic proteins in plants by Liuyi Dang; Els J.M. Van Damme (51-64).
Plants produce a broad spectrum of toxic proteins that confer resistance against herbivores and pathogens. An overview of different types of toxic proteins expressed in plants is presented and their biotechnological applications are discussed.Display OmittedPlants have evolved to synthesize a variety of noxious compounds to cope with unfavorable circumstances, among which a large group of toxic proteins that play a critical role in plant defense against predators and microbes. Up to now, a wide range of harmful proteins have been discovered in different plants, including lectins, ribosome-inactivating proteins, protease inhibitors, ureases, arcelins, antimicrobial peptides and pore-forming toxins.To fulfill their role in plant defense, these proteins exhibit various degrees of toxicity towards animals, insects, bacteria or fungi. Numerous studies have been carried out to investigate the toxic effects and mode of action of these plant proteins in order to explore their possible applications. Indeed, because of their biological activities, toxic plant proteins are also considered as potentially useful tools in crop protection and in biomedical applications, such as cancer treatment. Genes encoding toxic plant proteins have been introduced into crop genomes using genetic engineering technology in order to increase the plant’s resistance against pathogens and diseases. Despite the availability of ample information on toxic plant proteins, very few publications have attempted to summarize the research progress made during the last decades. This review focuses on the diversity of toxic plant proteins in view of their toxicity as well as their mode of action. Furthermore, an outlook towards the biological role(s) of these proteins and their potential applications is discussed.
Keywords: Plant protein; Toxicity; Biological activity; Mode of action; Plant defense; Biotechnological application;

Purification, sequencing and characterization of phospholipase D from Indian mustard seeds by Hafeeza Khatoon; Johanna Mansfeld; Angelika Schierhorn; Renate Ulbrich-Hofmann (65-75).
Phospholipase D from Indian mustard seeds was purified to homogeneity. Amino acid sequence data and enzymological properties suggest its assignment to α-type phospholipases DDisplay OmittedPhospholipase D (PLD; E.C. 3.1.4.4) is widespread in plants where it fulfills diverse functions in growth and in the response to stresses. The enzyme occurs in multiple forms that differ in their biochemical properties. In the present paper PLD from medicinally relevant Indian mustard seeds was purified by Ca2+-mediated hydrophobic interaction and anion exchange chromatography to electrophoretic homogeneity. Based on mass-spectrometric sequence analysis of tryptic protein fragments, oligonucleotide primers for cloning genomic DNA fragments that encoded the enzyme were designed and used to derive the complete amino acid sequence of this PLD. The sequence data, as well as the molecular properties (molecular mass of 92.0 kDa, pI 5.39, maximum activity at pH 5.5–6.0 and Ca2+ ion concentrations ⩾60 mM), allowed the assignment of this enzyme to the class of α-type PLDs. The apparent kinetic parameters Vmax and Km , determined for the hydrolysis of phosphatidylcholine (PC) in an aqueous mixed-micellar system were 356 ± 15 μmol min−1  mg−1 and 1.84 ± 0.17 mM, respectively. Phosphate analogs such as NaAlF4 and Na3VO4 displayed strong inhibition of the enzyme. Phosphatidylinositol 4,5-bisphosphate had a strong activating effect at 2–10 mM CaCl2. PLD was inactivated at temperatures >45 °C. The enzyme exhibited the highest activity toward PC followed by phosphatidylethanolamine and phosphatidylglycerol. PCs with short-chain fatty acids were better substrates than PCs with long fatty acid chains. Lyso-PC was not accepted as substrate.
Keywords: Phospholipase D; Brassica juncea; Purification; Amino acid sequence; Phosphatidylinositol 4,5-bisphosphate; Substrate specificity; Calcium; Inhibitors;

Low temperature tolerance in plants: Changes at the protein level by Mohsen Janmohammadi; Lello Zolla; Sara Rinalducci (76-89).
Low temperature (LT) is a major environmental restriction on plant performance. Crop plant acclimation to LT stress is mediated via intense proteomic changes affecting photosynthesis, defence/detoxification, signal transduction and metabolic pathways. The identification of LT-responsive proteins may provide the required backgrounds of plant genetic engineering.Display OmittedLow temperature (LT) is one of several important environmental stresses influencing plant performance and distribution. Adaptation to LT is a highly dynamic stress-response phenomenon and involves complex cross-talk between different regulatory levels. Although plants differ in their sensitivity to LT, in temperate species low nonfreezing temperatures cause noticeable alterations in various biochemical and physiological processes that can potentially improve freezing tolerance. This adaptation is associated with changes in the expression pattern of genes and their protein products. Proteins are the major players in most cellular events and are directly involved in plant LT responses, thereby proteome analysis could help uncover additional novel proteins associated with LT tolerance. Proteomics is recommended as an appropriate strategy for complementing transcriptome level changes and characterizing translational and post-translational regulations. In this review, we considered alterations in the expression and accumulation of proteins in response to LT stress in the three major cereal crops produced worldwide (wheat, barley, and rice). LT stress down-regulates many photosynthesis-related proteins. On the contrary, pathways/protein sets that are up-regulated by LT include carbohydrate metabolism (ATP formation), ROS scavenging, redox adjustment, cell wall remodelling, cytoskeletal rearrangements, cryoprotection, defence/detoxification. These modifications are common adaptation reactions also observed in the plant model Arabidopsis, thus representing key potential biomarkers and critical intervention points for improving LT tolerance of crop plants in cold regions with short summers. We believe that an assessment of the proteome within a broad time frame and during the different phenological stages may disclose the molecular mechanisms related to the developmental regulation of LT tolerance and facilitate the progress of genetically engineered stress-resistant plant varieties.
Keywords: Acclimation; LT-responsive proteins; Physiological changes; Proteome;

The willow PAL family consists of 5 genes encoding 4 isozymes that have higher kinetic activity, and a wider gene expression pattern, than that found for the closely related poplar PAL family.Display OmittedWillow is an important biomass crop for the bioenergy industry, and therefore optimal growth with minimal effects of biotic and abiotic stress is essential. The phenylpropanoid pathway is responsible for the biosynthesis of not only lignin but also of flavonoids, condensed tannins, benzenoids and phenolic glycosides which all have a role in protecting the plant against biotic and abiotic stress. All products of the phenylpropanoid pathway are important for the healthy growth of short rotation cropping species such as willow. However, the phenylpropanoid pathway in willow remains largely uncharacterised. In the current study we identified and characterised five willow phenylalanine ammonia-lyase (PAL) genes, which encode enzymes that catalyse the deamination of l-phenylalanine to form trans-cinnamic acid, the entry point into the phenylpropanoid pathway. Willow PAL1, PAL2, PAL3 and PAL4 genes were orthologous to the poplar genes. However no orthologue of PAL5 appears to be present in willow. Moreover, two tandemly repeated PAL2 orthologues were identified in a single contig. Willow PALs show similar sub-cellular localisation to the poplar genes. However, the enzyme kinetics and gene expression of the willow PAL genes differed slightly, with willow PAL2 being more widely expressed than its poplar orthologues implying a wider role for PALs in the production of flavonoids, condensed tannins, benzenoids, and phenolic glycosides, in willow.
Keywords: l-Phenylalanine ammonia-lyase (PAL); Salix viminalis (willow); Subcellular localisation; Enzyme kinetics; Gene expression; Phenylpropanoid metabolism; Gene family;

Flavonoids from Piper delineatum modulate quorum-sensing-regulated phenotypes in Vibrio harveyi by Alberto J. Martín-Rodríguez; Juan C. Ticona; Ignacio A. Jiménez; Ninoska Flores; José J. Fernández; Isabel L. Bazzocchi (98-106).
A series of natural flavonoids from Piper delineatum exhibited the ability to disrupt QS-mediated bioluminescence downstream LuxO and showed a strong dose-dependent inhibition of biofilm formation, in the bacterial model Vibrio harveyi.Display OmittedQuorum sensing (QS), or bacterial cell-to-cell communication, is a key process for bacterial colonization of substrata through biofilm formation, infections, and production of virulence factors. In an ongoing investigation of bioactive secondary metabolites from Piper species, four new flavonoids (14), along with five known ones (59) were isolated from the leaves of Piper delineatum. Their stereostructures were established by spectroscopic and spectrometric methods, including 1D and 2D NMR experiments, and comparison with data reported in the literature. The compounds were screened for their ability to interfere with QS signaling in the bacterial model Vibrio harveyi. Four compounds from this series (2, 3, 6, and 7) exhibited remarkable activity in the micromolar range, being compounds 3 and 7 particularly attractive since they did not affect bacterial growth. The results suggest that these flavonoids disrupt QS-mediated bioluminescence by interaction with elements downstream LuxO in the QS circuit of V. harveyi, and also, they exhibited a strong dose-dependent inhibition of biofilm formation. The present findings shed light on the QS inhibition mechanisms of flavonoids, underlining their potential applications.
Keywords: Piper delineatum; Flavonoids; Quorum sensing; Biofilm; Vibrio harveyi;

Grayanane and leucothane diterpenoids from the leaves of Rhododendron micranthum by Mengke Zhang; Yangyang Xie; Guanqun Zhan; Liang Lei; Penghua Shu; Yongli Chen; Yongbo Xue; Zengwei Luo; Qian Wan; Guangmin Yao; Yonghui Zhang (107-115).
Twelve previously unknown and eleven known diterpenoids were isolated from the leaves of Rhododendron micranthum. Four of their structures were confirmed by single-crystal X-ray diffraction. Their in vitro immunomodulatory activities were evaluated.Display OmittedEleven grayanane diterpenoids, 1-epi-grayanotoxin IV, 1-epi-grayanotoxin II, 6-deoxy-1-epi-grayanotoxin XVII, 6-deoxygrayanotoxin XVII, 16-acetylgrayanotoxin II, 3-oxograyanotoxin IX, 14-deoxygrayanotoxin VIII, 14-acetylisograyanotoxin II, rhodomicranols C–E, and a leucothane diterpenoid, rhodomicranol F, together with eleven known diterpenoids were isolated from leaves of Rhododendron micranthum. Their structures were elucidated by spectroscopic analyses, with the absolute configurations of 1-epi-grayanotoxin IV and rhodomicranol C determined by single-crystal X-ray diffraction with Cu Kα radiation, and the structures of 14-acetylisograyanotoxin II and known grayanotoxins IX and X confirmed by single-crystal X-ray diffraction. All twenty-three diterpenoids were evaluated for their in vitro immunomodulatory activities, and none showed significant immunomodulatory activities in a dose-dependent manner. In addition, they are non-toxic to the murine lymphocytes in the general cytotoxicity assay.
Keywords: Rhododendron micranthum; Ericaceae; Grayanane diterpenoids; Leucothane diterpenoids; Absolute configuration; Single-crystal X-ray diffraction; Immunomodulatory activity;

Botryane, noreudesmane and abietane terpenoids from the ascomycete Hypoxylon rickii by Eric Kuhnert; Frank Surup; Vincent Wiebach; Steffen Bernecker; Marc Stadler (116-122).
Ten terpenoids, among those seven novel natural products, were isolated from cultures of the tropical ascomycete Hypoxylon rickii (Xylariaceae). Their structures were elucidated using 2D NMR and HRESIMS. The new compounds belong to the botryane, 14-noreudesmane and abietane types. Compounds 1, 2, 3, 7, and 10 showed cytotoxic effects against murine cells.Display OmittedIn the course of our screening for new bioactive natural products, a culture of Hypoxylon rickii, a xylariaceous ascomycete collected from the Caribbean island Martinique, was identified as extraordinary prolific producer of secondary metabolites. Ten metabolites of terpenoid origin were isolated from submerged cultures of this species by preparative HPLC. Their structures were elucidated using spectral techniques including 2D NMR and HRESIMS. Three of the compounds were elucidated as new botryanes (13) along with three known ones, i.e. (3aS)-3a,5,5,8-tetramethyl-3,3a,4,5-tetrahydro-1H-cyclopenta[de]isochromen-1-one (4), (3aS,8R)-3a,5,5,8-tetramethyl-3,3a,4,5,7,8-hexahydro-1H-cyclopenta[de]isochromen-1-one (5) and botryenanol (6). Further three new sesquiterpenoids featured a 14-noreudesmane-type skeleton and were named hypoxylan A–C (79); the diterpenoid rickitin A (10) contains an abietane-type backbone. Compounds 1, 2, 3, 7, and 10 showed cytotoxic effects against murine cells.
Keywords: Hypoxylon; Xylariaceae; Abietane; Botryane; Eudesmane; Terpenes; Secondary metabolites; Structure elucidation; Ascomycetes; Biodiversity; Endophytes; Terpenoids; Xylariales;

Anti-viral and cytotoxic norbisabolane sesquiterpenoid glycosides from Phyllanthus emblica and their absolute configurations by Jun-Jiang Lv; Shan Yu; Ying Xin; Rong-Rong Cheng; Hong-Tao Zhu; Dong Wang; Chong-Ren Yang; Min Xu; Ying-Jun Zhang (123-134).
Pre-column derivative/chiral separation/DAD detection and theoretical calculations of ECD spectra using TDDFT were used to determine the absolute configurations of phyllaemblicins H1–H14, with antiviral activities, isolated from P. emblica.Display OmittedIn an effort to identify anti-viral and cytotoxic compounds from Phyllanthus spp., 14 highly oxygenated norbisabolane sesquiterpenoids, phyllaemblicins H1–H14, were isolated from the roots of Phyllanthus emblica Linn, along with phyllaemblicins B and C and glochicoccinoside D. Their structures were determined on the basis of detailed spectroscopic analysis and chemical methods. Determination of absolute configurations of these compounds was facilitated by theoretical calculations of electronic circular dichroism (ECD) spectra using time-dependent density functional theory (TDDFT) for the aglycone components, and pre-column derivative/chiral HPLC analysis for the monosaccharides. The known glochicoccinoside D displayed potent activity against influenza A virus strain H3N2 and hand, foot and mouth virus EV71, with IC50 values of 4.5 ± 0.6 and 2.6 ± 0.7 μg/ml, respectively. Phyllaemblicin H1 showed moderate cytotoxicity against human cancer cell lines A-549 and SMMC-7721, with IC50 values of 4.7 ± 0.7 and 9.9 ± 1.3 μM, respectively.
Keywords: Yu Gan Zi; Phyllanthus emblica; Euphorbiaceae; ECD calculation; Antiviral; Norbisabolane sesquiterpenoid glycosides; Phyllaemblicins;

Chemical constituents of Abies fabri by Yong-Li Li; Yan-Xia Gao; Hui-Zi Jin; Lei Shan; Wan-Lin Chang; Xian-Wen Yang; Hua-Wu Zeng; Ning Wang; Andre Steinmetz; Wei-Dong Zhang (135-143).
Twelve previously unreported compounds and 80 known ones were isolated from Abies fabri. Their structures were elucidated mainly by spectroscopic methods. The absolute configurations of compounds 1, 3, and 7 were determined by Cu-Kα X-ray crystallographic analysis. One known compound, 23-hydroxy-3-oxolanosta-8,24-dien-26,23-olide, showed weak cytotoxic activity against A549 and THP-1 cells.Display OmittedSystematic phytochemical investigations on Abies fabri resulted in the isolation of 94 compounds, consisting of 68 terpenoids, six lignans, seven flavonoids, and 13 other miscellaneous chemical constituents. Their structures were elucidated on the basis of spectroscopic methods, and the absolute configurations of three of these previously unknown compounds were determined by Cu-Kα X-ray crystallographic analysis. Twelve previously unreported compounds, one artifact, and one potential artifact were identified, including six triterpenoids, four diterpenoids, two sesquiterpenoids, one lignan, and one phenol. 23-Hydroxy-3-oxolanosta-8,24-dien-26,23-olide showed weak cytotoxic activity against A549 and THP-1 cells with the IC50 values of 5.3 and 5.1 μM, respectively.
Keywords: Abies fabri; Pinaceae; Triterpenoids; Diterpenoids; Cytotoxicity; Cu-Kα X-ray crystallography;

Prevalence and specificity of Baeyer–Villiger monooxygenases in fungi by Lorena Butinar; Martina Mohorčič; Valérie Deyris; Katia Duquesne; Gilles Iacazio; Magalie Claeys-Bruno; Josepha Friedrich; Véronique Alphand (144-153).
A screening of a hundred fungi from three phyla revealed that Baeyer–Villiger monooxygenases (BVMO) are quasi-ubiquitous enzymes. Statistical analyses showed that bicyclo[3.2.0]heptenone biotransformation profiles are partly related to the taxonomic classification and suggest that the BVMOs from Zygomycota present original features.Display OmittedOut of 107 fungal strains belonging to three phyla (Ascomycota, Basidiomycota and Zygomycota) and 46 genera, 86 exhibited Baeyer–Villiger monooxygenase (BVMO) activity against racemic bicyclo[3.2.0]heptenone. The strains were classified into three “profiles” based on regio- and enantioselectivity. Statistical analyses of our results, extended by literature data, showed that these profiles could be related to the taxonomic classification of the strains, and suggest that the BVMOs from the Zygomycota phylum may be different in their primary structures from established ones.
Keywords: Baeyer–Villiger monooxygenase; Screening; Fungi; Phylum; Ascomycetes; Basidiomycetes; Zygomycetes; Regioselectivity; Enantioselectivity;

A novel form of ficin from Ficus carica latex: Purification and characterization by Danielle Baeyens-Volant; André Matagne; Rachida El Mahyaoui; Ruddy Wattiez; Mohamed Azarkan (154-167).
A novel ficin form was isolated from Ficus carica latex by combination of cation-exchange, hydrophobic interaction, affinity and fplc-gel filtration chromatographies. Mass spectrometry and far-UV CD analyses revealed very close molecular mass and secondary structure contents comparable to the known ficins. N-terminal sequencing and gel electrophoresis clearly demonstrated a novel protease form. Enzymatic activity measurements suggested different substrate specificities. The novel ficin resembles a papain-like protease according to inhibitory activity data.Display OmittedA novel ficin form, named ficin E, was purified from fig tree latex by a combination of cation-exchange chromatography on SP-Sepharose Fast Flow, Thiopropyl Sepharose 4B and fplc-gel filtration chromatography. The new ficin appeared not to be sensitive to thiol derivatization by a polyethylene glycol derivative, allowing its purification. The protease is homogeneous according to PAGE, SDS–PAGE, mass spectrometry, N-terminal micro-sequencing analyses and E-64 active site titration. N-terminal sequencing of the first ten residues has shown high identity with the other known ficin (iso)forms. The molecular weight was found to be (24,294 ± 10) Da by mass spectrometry, a lower value than the apparent molecular weight observed on SDS–PAGE, around 27 kDa. Far-UV CD data revealed a secondary structure content of 22% α-helix and 26% β-sheet. The protein is not glycosylated as shown by carbohydrate analysis. pH and temperature measurements indicated maxima activity at pH 6.0 and 50 °C, respectively. Preliminary pH stability analyses have shown that the protease conserved its compact structure in slightly acidic, neutral and alkaline media but at acidic pH (<3), the formation of some relaxed or molten state was evidenced by 8-anilino-1-naphtalenesulfonic acid binding characteristics. Comparison with the known ficins A, B, C, D1 and D2 (iso)forms revealed that ficin E showed activity profile that looked like ficin A against two chromogenic substrates while it resembled ficins D1 and D2 against three fluorogenic substrates. Enzymatic activity of ficin E was not affected by Mg2+, Ca2+ and Mn2+ at a concentration up to 10 mM. However, the activity was completely suppressed by Zn2+ at a concentration of 1 mM. Inhibitory activity measurements clearly identified the enzyme as a cysteine protease, being unaffected by synthetic (Pefabloc SC, benzamidine) and by natural proteinaceous (aprotinin) serine proteases inhibitors, by aspartic proteases inhibitors (pepstatin A) and by metallo-proteases inhibitors (EDTA, EGTA). Surprisingly, it was well affected by the metallo-protease inhibitor o-phenanthroline. The enzymatic activity was however completely blocked by cysteine proteases inhibitors (E-64, iodoacetamide), by thiol-blocking compounds (HgCl2) and by cysteine/serine proteases inhibitors (TLCK and TPCK). This is a novel ficin form according to peptide mass fingerprint analysis, specific amidase activity, SDS–PAGE and PAGE electrophoretic mobility, N-terminal sequencing and unproneness to thiol pegylation.
Keywords: Ficin; Ficus carica; Cysteine protease; Latex; Mass spectrometry; Circular dichroism;

Isolation and extraction of ruberythric acid from Rubia tinctorum L. and crystal structure elucidation by Lauren Ford; Christopher M. Rayner; Richard S. Blackburn (168-173).
Ruberythric acid is extracted from madder (Rubia tinctorum L.) and characterised using a variety of techniques including 1H and 13C NMR. The first known X-ray crystal structure of ruberythric acid is presented.Display OmittedMadder (Rubia tinctorum L.) has been exploited as a dye throughout history. The roots of the plant are very rich in the highly coloured glycosidic compounds ruberythric acid and lucidin primeveroside, alongside the corresponding aglycons which can be readily formed by deglycosylation, particularly during extraction. Supported by 1H and 13C NMR data, the conclusive X-ray crystal structure of the natural dye ruberythric acid is presented for the first time. The solid state structure revealed extensive intermolecular hydrogen bonding interactions between the sugar moieties in the unit cell, but only intramolecular hydrogen bonding through the hydroxyquinone groups. There is also some additional π–π stacking from the anthraquinone moiety.
Keywords: Madder; Rubia tinctorum; Dye; Alizarin; Purpurin; Ruberythric acid; Anthraquinone glycoside; Extraction; X-ray crystal structure; Adsorption mechanism;

Changes in gene transcription and taxane production in elicited cell cultures of Taxus  ×  media and Taxus globosa by Karla Ramirez-Estrada; Lidia Osuna; Elisabeth Moyano; Mercedes Bonfill; Nadia Tapia; Rosa M. Cusido; Javier Palazon (174-184).
Response to elicitation in Taxus cell cultures varies according to the plant species, resulting in changes in the transcription profiles of taxane biosynthetic genes and an enhanced taxane production.Display OmittedThe response of two Taxus cell systems to the action of cyclodextrin (CD) and coronatine (CORO), supplied to the culture medium either separately or together, was studied. Two-stage Taxus globosa and Taxus media cell cultures were established and the elicitors were added at the beginning of the second stage. Growth, taxane production, and the expression of known taxol biosynthetic genes, including the recently characterized CoA ligase gene, were studied. Although CORO reduced the growth capacity of both cell lines, CD apparently counteracted this negative effect. Taxane production was significantly enhanced by the simultaneous addition of CD and CORO to the medium. The total taxane production in the T. media cell line was more than double that of T. globosa, but in the latter more than 90% of the taxanes produced were excreted to the medium. Individual taxane patterns also differed: at the height of production, the main taxanes in T. globosa cultures were cephalomannine and 10-deacetyltaxol, and in T. media, taxol and baccatin III. The low transcript levels of taxane biosynthetic genes found in T. globosa cells mirrored the lower taxane production in these cultures, while a high expression was strongly correlated with a high taxane production in T. media.
Keywords: Plant cell cultures; Taxus globosa; Taxus  ×  media; Taxanes; Taxol; Transcription profiles;

Phenylethanoid glycosides in tepals of Magnolia salicifolia and their occurrence in flowers of Magnoliaceae by Elaine A. Porter; Geoffrey C. Kite; Nigel C. Veitch; Ivey A. Geoghegan; Sonny Larsson; Monique S.J. Simmonds (185-193).
Yulanosides A and B and 2′-rhamnoechinacoside were characterised from tepals of Magnolia salicifolia together with the known phenylethanol glycosides crassifolioside, echinacoside and verbascoside. Yulanosides are characteristic of Magnolia subgenus Yulania.Phenylethanoid glycosides were among the major UV-absorbing components in 80% aq. CH3OH extracts of the tepals of Magnolia salicifolia (Siebold & Zucc.) Maxim. (Magnoliaceae; Magnolia subgenus Yulania). Structural characterisation of isolated compounds by spectroscopic and chemical methods revealed three previously unrecorded examples, yulanoside A, yulanoside B and 2′-rhamnoechinacoside, and the known compounds echinacoside and crassifolioside; chromatographic methods also identified verbascoside in the tepal extract. Yulanoside A is the first reported example of a phenylethanoid pentaglycoside, namely hydroxytyrosol 1-O-{β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranosyl-(1 → 6)-[3,4-dihydroxycinnamoyl-(→ 4)][α-l-rhamnopyranosyl-(1 → 3)][α-l-rhamnopyranosyl-(1 → 2)]-β-d-glucopyranoside}. A survey of Magnolia sensu lato and Liriodendron (the two genera of Magnoliaceae) suggested that yulanoside A and its deglucosyl derivative (yulanoside B) were a feature of the tepal chemistry of Magnolia subgenus Yulania (except Magnolia acuminata, the sole member of section Tulipastrum, which did not accumulate phenylethanoid glycosides). The two species of Liriodendron and examined examples of Magnolia subgenus Magnolia sections Magnolia and Rytidospermum (subsection Oyama) also accumulated phenylethanoid glycosides in their tepals and in these species, and in subgenus Yulania, the major compounds were one or more of echinacoside, 2′-rhamnoechinacoside, crassifolioside and verbascoside. Levels of phenylethanoid glycosides were found to be much lower in species studied from Magnolia sections Gwillimia, Macrophylla and Rytidospermum (subsection Rytidospermum), although yulanoside A was detectable in M. macrophylla and this may have some bearing on the placement of section Macrophylla, which is currently uncertain. In the isolates of yulanoside B and echinacoside, minor phenylethanoid glycosides were determined to be analogues of these compounds with β-d-xylose at C-3′ of the primary glucose rather than α-l-rhamnose.
Keywords: Magnolia salicifolia; Liriodendron; Magnoliaceae; Flower; Chemosystematics; Phenylethanoid glycosides; Yulanoside A;

Minor oxygenated cannabinoids from high potency Cannabis sativa L. by Safwat A. Ahmed; Samir A. Ross; Desmond Slade; Mohamed M. Radwan; Ikhlas A. Khan; Mahmoud A. ElSohly (194-199).
Nine oxygenated cannabinoids (19) were isolated from a high potency Cannabis sativa L. variety. Their chemical structures were elucidated using spectroscopic techniques. The antimicrobial, antileishmanial and antimalarial activities were tested.Display OmittedNine oxygenated cannabinoids were isolated from a high potency Cannabis sativa L. variety. Structure elucidation was achieved using spectroscopic techniques, including 1D and 2D NMR, HRMS and GC–MS. These minor compounds include four hexahydrocannabinols, four tetrahydrocannabinols, and one hydroxylated cannabinol, namely 9α-hydroxyhexahydrocannabinol, 7-oxo-9α-hydroxyhexa-hydrocannabinol, 10α-hydroxyhexahydrocannabinol, 10aR-hydroxyhexahydrocannabinol, Δ9-THC aldehyde A, 8-oxo-Δ9-THC, 10aα-hydroxy-10-oxo-Δ8-THC, 9α-hydroxy-10-oxo-Δ6a,10a-THC, and 1′S-hydroxycannabinol, respectively. The latter compound showed moderate anti-MRSa (IC50 10.0 μg/mL), moderate antileishmanial (IC50 14.0 μg/mL) and mild antimalarial activity against Plasmodium falciparum (D6 clone) and P. falciparum (W2 clone) with IC50 values of 3.4 and 2.3 μg/mL, respectively.
Keywords: Cananbis; Cannabis sativa; Cannabaceae; High potency; Oxygenated cannabinoids; Anti-bacterial; Anti-leishmanial; Anti-malarial;

A series of new phloroglucinols were identified from two distinct Cystophora species using HPLC–NMR & HPLC–MS in conjunction with an off-line isolation approach.Display OmittedThe crude dichloromethane extracts of the marine brown algae Cystophora monilifera and Cystophora subfarcinata were subjected to phytochemical profiling. This enabled the structures of both new and known phloroglucinols to be dereplicated and proposed using HPLC–NMR and HPLC–MS. Subsequent isolation confirmed the presence of four new and eight previously reported compounds. Five of the isolated phloroglucinols displayed selective antimicrobial activity.
Keywords: Cystophora; Brown alga; Phloroglucinol; HPLC–NMR; HPLC–MS; Dereplication; Antimicrobial activity;

Partial depolymerization of genetically modified potato tuber periderm reveals intermolecular linkages in suberin polyester by José Graça; Vanessa Cabral; Sara Santos; Pedro Lamosa; Olga Serra; Marisa Molinas; Lukas Schreiber; Friedrich Kauder; Rochus Franke (209-219).
Potato periderm suberin builds as a macromolecule from a glycerol – octadec-9-ene-1,18-dioic acid – glycerol backbone, linked in its periphery to polyaromatics through the glycerol – 18-hydroxyoctadec-9-enoic acid – ferulic acid connection.Display OmittedSuberin is a biopolyester found in specialized plant tissues, both internal and external, with key frontier physiological functions. The information gathered so far from its monomer and oligomer composition, and in situ studies made by solid state techniques, haven’t solved the enigma of how the suberin polyester is assembled as a macromolecule. To investigate how monomers are linked in suberin, we analyzed oligomer fragments solubilized by the partial depolymerization of suberin from potato (Solanum tuberosum) tuber periderms. The structure of the suberin oligomers, namely which monomers they included, and the type and frequency of the inter-monomer ester linkages, was assessed by ESI-MS/MS and high resolution NMR analysis. The analyzed potato periderms included the one from wild type (cv. Desirée) and from plants where suberin-biosynthesis genes were downregulated in chain elongation (StKCS6), ω-hydroxylation (CYP86A33) and feruloylation (FHT).Two building blocks were identified as possible key structures in the macromolecular development of the potato periderm suberin: glycerol – α,ω-diacid – glycerol, as the core of a continuous suberin aliphatic polyester; and glycerol – ω-hydroxyacid – ferulic acid, anchoring this polyaliphatic matrix at its periphery to the vicinal polyaromatics, through linking to ferulic acid. The silencing of the StKCS6 gene led to non-significant alterations in suberin structure, showing the relatively minor role of the very-long chain (>C28) fatty acids in potato suberin composition. The silencing of CYP86A33 gene impaired significantly suberin production and disrupted the biosynthesis of acylglycerol structures, proving the relevance of the latter and thus of the glycerol – α,ω-diacid – glycerol unit for the typical suberin lamellar organization. The silencing of the FHT gene led to a lower frequency of ferulate linkages in suberin polyester but to more polyphenolic guaiacyl units as seen by FTIR analyses in the intact polymer.
Keywords: Solanum tuberosum; Solanaceae; Potato periderm; Suberin; Acylglycerols of α,ω-diacids; Ferulates of ω-hydroxyacids; CYP86A33 ω-hydroxylase; FHT feruloyl transferase; StKCS6 fatty acid chain elongase; Partial depolymerization;

Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening by Dimitra Chormova; Lenka Franková; Andrew Defries; Sean R. Cutler; Stephen C. Fry (220-236).
Xenobiotics that inhibit the cell-wall-remodelling enzyme activity, xyloglucan endotransglucosylase (XET), would be a valuable tool in ‘chemical genetics’ for elucidating its biological roles. By screening 4216 small molecules in a dot-blot assay on paper-immobilised xyloglucan, we discovered 45 compounds that inhibited or promoted XET activity. The most effective inhibitors were singlet oxygen generators.Display OmittedSmall molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes’ biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme–cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors – especially compounds that generate singlet oxygen (1O2) e.g., riboflavin (IC50 29 μM), retinoic acid, eosin (IC50 27 μM) and erythrosin (IC50 36 μM). The riboflavin effect was light-dependent, supporting 1O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (−)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1 μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40 μM mycophenolic acid and (−)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads.
Keywords: Anthraquinones; Cell wall; Chemical genetics; Dot-blot assay; Flavonoids; Riboflavin; Singlet oxygen; Sulphydryl reagents; Tannins; Xyloglucan endotransglucosylase (XET);

Prenylated flavanone derivatives isolated from Erythrina addisoniae are potent inducers of apoptotic cell death by Claus M. Passreiter; Anke-Katrin Suckow-Schnitker; Andreas Kulawik; Jonathan Addae-Kyereme; Colin W. Wright; Wim Wätjen (237-244).
The prenylated flavanones addisoniaflavanones I and II isolated from Erythrina addisoniae stem bark possess a cytotoxic and pro-apoptotic potential (caspase-3/7 activation, nuclear fragmentation) in H4IIE hepatoma cells.Display OmittedExtracts of Erythrina addisoniae are frequently used in the traditional medicine of Western Africa, but insufficient information about active compounds is available. From the stem bark of E. addisoniae, three (1, 2, 4) and three known (3, 5, 6) flavanones were isolated: addisoniaflavanones I and II, containing either a 2″,3″-epoxyprenyl moiety (1) or a 2″,3″-dihydroxyprenyl moiety (2) were shown to be highly toxic (MTT assay: EC50 values of 5.25 ± 0.7 and 8.5 ± 1.3 μM, respectively) to H4IIE hepatoma cells. The cytotoxic potential of the other isolated flavanones was weaker (range of EC50 values between 15 and >100 μM). Toxic effects of addisoniaflavanone I and II were detectable after 3 h (MTT assay). Both compounds induced an apoptotic cell death (caspase-3/7 activation, nuclear fragmentation) in the hepatoma cells and, at high concentrations, also necrosis (membrane disruption: ethidium bromide staining). Formation of DNA strand breaks was not detectable after incubation with these compounds (comet assay). In conclusion, the prenylated flavanones addisoniaflavanones I and II may be of interest for pharmacological purposes due to their high cytotoxic and pro-apoptotic potential against hepatoma cells.
Keywords: Apoptosis; Bioactivity; Cytotoxicity; Flavonoid; Prenylation;

Sesquiterpenes, flavonoids, shikimic acid derivatives and pyrrolizidine alkaloids from Senecio kingii Hook by Liliana Ruiz-Vásquez; Matías Reina; M. López-Rodríguez; Cristina Giménez; Raimundo Cabrera; Pedro Cuadra; Víctor Fajardo; Azucena González-Coloma (245-253).
Display OmittedTwenty-four compounds including eleven eremophilanolides (111), one eremophilane (13), five shikimic acid derivatives (1418), six flavonoids (1924), and the macrocyclic unsaturated pyrrolizidine alkaloid integerrimine (25) were isolated from Senecio kingii, an endemic species from the Magallanes Region (Chile). Compounds 3, 5, 6, 811 and 1318 have not been previously reported as natural products. Their molecular structures were determined by NMR spectroscopic analysis and comparison with published NMR data. An X-ray-analysis of compound 3 has been performed. Their insecticidal and antifungal activities were tested, being compound 3 the strongest insect antifeedant. Compounds 6, 9 and 18 were moderate antifungals.
Keywords: Asteraceae; Senecio kingii; Eremophilanolides; Shikimic acid derivatives; Flavonoids; Pyrrolizidine alkaloids; Structure–activity;

Novel stilbenoids, including cannabispiradienone glycosides, from Tragopogon tommasinii (Asteraceae, Cichorieae) and their potential anti-inflammatory activity by Sebastian Granica; Jakub P. Piwowarski; Antonio Randazzo; Peter Schneider; Barbara Żyżyńska-Granica; Christian Zidorn (254-266).
Aerial parts of Tragopogon tommasinii Sch.Bip. (Asteraceae, Cichorieae) yielded 21 natural compounds, including seven new stilbenoids; three simple bibenzyls, two benzylphthalides, and two glucopyranosyl-de-methoxy-canna-bispiradienoides. Structures were established by HR mass spectrometry and extensive 1D and 2D NMR spectroscopy. Bioactivity of new compounds on human neutrophils and on their release of IL-8, IL-1β TNF-α, and MMP-9 and on the surface expression of TLR-4 in particular was investigated.Display OmittedA phytochemical investigation of Tragopogon tommasinii Sch.Bip. (Asteraceae, Cichorieae) yielded a total of 21 natural products, two simple phenolic acids (4-hydroxybenzoic acid and p-coumaric acid), four caffeic acid derivatives (chlorogenic acid, 3-O-caffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid), six flavonoids (luteolin, luteolin 7-O-glucoside, vitexin, orientin, quercetin 3-O-glucoside, and isorhamnetin 3-O-glucoside), three simple bibenzyls [2-carboxyl-5-hydroxy-3-methoxy-4′-β-glucopyranosyl-oxybibenzyl, 3-caffeoyl-(9 → 5)-β-apiosyl-(1 → 6)-β-glucopyranosyloxy-5,4′-dihydroxy-3′-methoxybibenzyl, 3-caffeoyl-(9 → 5)-β-apiosyl-(1 → 6)-β-glucopyranosyloxy-4′-dihydroxy-5,3′-dimethoxybibenzyl], three phtalides [3-(4-β-glucopyranosyloxybenzyl)-7-hydroxy-5-methoxyphtalide, 7-β-glucopyranosyloxy-(S)-3-(4-hydroxybenzyl)-5-methoxyphtalide, and 7-(1 → 6)-α-rhamnosyl-β-glucopyranosyloxy-(S)-3-(4-hydroxybenzyl)-5-methoxyphtalide], two cannabispiradienone derivatives [3-O-β-glucopyranosyldemethoxycannabispiradienone and 3-caffeoyl-(9 → 5)-β-apiosyl-(1 → 6)-β-glucopyranosyloxydemethoxycannabispiradienone], and tetra-N-coumaroyl spermine. The three bibenzyls, the latter two benzylphthalides, and both cannabispiradienone derivatives represent new natural compounds and all compounds, except the caffeic acid derivatives and the flavonoids were new for T. tommasinii. The structures were established by HR mass spectrometry, extensive 1D and 2D NMR spectroscopy, and CD spectroscopy. Moreover, the potential anti-inflammatory activities of the new compounds were assayed using human neutrophils and their production of IL-1b, IL-8, TNF-α and MMP-9 as well as the expression of TLR-4, respectively.
Keywords: Tragopogon; Asteraceae; Tribe Cichorieae; Subtribe Scorzonerinae; Stilbenoids;

Plant betalains: Chemistry and biochemistry by Mohammad Imtiyaj Khan; P. Giridhar (267-295).
This review discusses all identified betalains from plant sources with relevant chemical information for easy reference. Annual production potential of betalains from plant sources has been computed first time in this review. Betalain and anthocyanin-accumulating families under Caryophyllales have been separately compiled. In addition, a comprehensive pathway for betalain biosynthesis is illustrated vis-a-vis their regulation.Display OmittedBetalains are vacuolar pigments composed of a nitrogenous core structure, betalamic acid [4-(2-oxoethylidene)-1,2,3,4-tetrahydropyridine-2,6-dicarboxylic acid]. Betalamic acid condenses with imino compounds (cyclo-l-3,4-dihydroxy-phenylalanine/its glucosyl derivatives), or amino acids/derivatives to form variety of betacyanins (violet) and betaxanthins (yellow), respectively. About 75 betalains have been structurally unambiguously identified from plants of about 17 families (known till date) out of 34 families under the order Caryophyllales, wherein they serve as chemosystematic markers. In this review, all the identified betalain structures are presented with relevant discussion. Also, an estimated annual production potential of betalains has been computed for the first time. In addition, mutual exclusiveness of anthocyanins and betalains has been discussed in the wake of new evidences. An inclusive list of betalain-accumulating plants reported so far has been presented here to highlight pigment occurrence and accumulation pattern. Betalain synthesis starts with hydroxylation of tyrosine to DOPA, and subsequent cleavage of aromatic ring of DOPA resulting to betalamic acid formation. This pathway consists of two key enzymes namely, bifunctional tyrosinase (hydroxylation and oxidation) and DOPA dioxygenase (O2-dependent aromatic ring cleavage). Various spontaneous cyclisation, condensation and glucosylation steps complement the extended pathway, which has been presented here comprehensively. The biosynthesis is affected by various ecophysiological factors including biotic and abiotic elicitors that can be manipulated to increase pigment production for commercial scale extraction. Betalains are completely safe to consume, and contribute to health.
Keywords: Betacyanins; Betaxanthins; Biosynthesis; Regulation; Ecophysiological factors;

Determination of free and glucosidically-bound volatiles in plants. Two case studies: L-menthol in peppermint (Mentha x piperita L.) and eugenol in clove (Syzygium aromaticum (L.) Merr. & L.M.Perry) by Barbara Sgorbini; Cecilia Cagliero; Alberto Pagani; Marla Sganzerla; Lorenzo Boggia; Carlo Bicchi; Patrizia Rubiolo (296-305).
The quali-quantitative analysis of L-menthol and eugenol in peppermint aerial parts and cloves has been obtained by the use of high-concentration-capacity sample preparation techniques (SBSE, and HS-SPME and in-solution SPME) in combination with GC-FID/MS.Display OmittedThis study arises from both the today’s trend towards exploiting plant resources exhaustively, and the wide quantitative discrepancy between the amounts of commercially-valuable markers in aromatic plants and those recovered from the related essential oil. The study addresses the determination of both the qualitative composition and the exhaustive distribution of free and glucosidically-bound L-menthol in peppermint aerial parts (Mentha x piperita L., Lamiaceae) and of eugenol in dried cloves (Syzygium aromaticum (L.) Merr. & L.M.Perry, Myrtaceae), two plants known to provide widely ranging essential oil yields. The two markers were investigated in essential oils and residual hydrodistillation waters, before and after enzymatic hydrolysis. Their amounts were related to those in the headspace taken as reference.The results showed that the difference between marker compound in headspace and in essential oil amounted to 22.8% for L-menthol in peppermint, and 16.5% for eugenol in cloves. The aglycones solubilised in the residual hydrodistillation waters were 7.2% of the headspace reference amount for L-menthol, and 13.3% for eugenol, respectively representing 9.3% and 15.9% of their amounts in the essential oil. The amount of L-menthol from its glucoside in residual hydrodistillation waters was 20.6% of that in the related essential oil, while eugenol from its glucoside accounted for 7.7% of the amount in clove essential oil. The yield of L-menthol, after submitting the plant material to enzymatic hydrolysis before hydrodistillation, increased by 23.1%, and for eugenol the increase was 8.1%, compared to the amount in the respective conventional essential oils.This study also aimed to evaluate the reliability of recently-introduced techniques that are little applied, if at all, in this field. The simultaneous use of high-concentration-capacity sample preparation techniques (SBSE, and HS-SPME and in-solution SPME) to run quali-quantitative analysis without sample manipulation, and direct LC–MS glucoside analysis, provided cross-validation of the results.
Keywords: Mentha x piperita L. (Lamiaceae); Syzygium aromaticum (L.) Merr. & L.M.Perry (Myrtaceae); Essential oils; Residual distillation waters; Monoterpene glucosides; Enzymatic hydrolysis; L-menthol; Eugenol; Quantitative determination;

Display Omitted Dendrobium nobile is an important medicinal orchid having profound importance in traditional herbal drug preparations and pharmacopeias worldwide. Due to various anthropogenic pressures the natural populations of this important orchid species are presently facing threats of extinction. In the present study, genetic and chemical diversity existing amongst 6 natural populations of D. nobile were assessed using molecular markers, and the influence of genetic factors on its phytochemical activity especially antioxidant potential was determined. Molecular fingerprinting of the orchid taxa was performed using ISSR and DAMD markers along with the estimation of total phenolics, flavonoids and alkaloid contents. Antioxidant activity was also measured using DPPH and FRAP assays which cumulatively revealed a significant level of variability across the sampled populations. The representatives from Sikkim in Northeast India revealed higher phytochemical activity whereas those from Mizoram showed lesser activity. Analysis of molecular variance (AMOVA) revealed that variation amongst the populations was significantly higher than within the populations. The data generated by UPGMA and Bayesian analytical models were compared in order to estimate the genetic relationships amongst the D. nobile germplasm sampled from different geographical areas of Northeast India. Interestingly, identical grouping patterns were exhibited by both the approaches. The results of the present study detected a high degree of existing genetic and phytochemical variation amongst the populations in relation to bioclimatic and geographic locations of populations. Our results strongly establish that the cumulative marker approach could be the best suited for assessing the genetic relationships with high accuracy amongst distinct D. nobile accessions.
Keywords: Dendrobium nobile; PIC; ISSR; DAMD; Genetic variation; Northeast India; Phytochemicals; Antioxidants; Medicinal orchids; Genetic diversity; Molecular barcoding; Medicinal plant;

Biologically active vallesamine, strychnan, and rhazinilam alkaloids from Alstonia: Pneumatophorine, a nor-secovallesamine with unusual incorporation of a 3-ethylpyridine moiety by Jun-Lee Lim; Kae-Shin Sim; Kien-Thai Yong; Bi-Juin Loong; Kang-Nee Ting; Siew-Huah Lim; Yun-Yee Low; Toh-Seok Kam (317-324).
Four alkaloids comprising two vallesamine, one strychnan, and pyranopyridine alkaloid were isolated from two Malayan Alstonia species, A. pneumatophora and A. rostrata. Several of the isolated alkaloids show antiproliferative (e.g., rhazinicine) and vasorelaxation (e.g., pneumatophorine, undulifoline) effects.Display OmittedFour alkaloids comprising two vallesamine, one strychnan, and one pyranopyridine alkaloid, in addition to 32 other known alkaloids were isolated from two Malayan Alstonia species, Alstonia pneumatophora and Alstonia rostrata. The structures of these alkaloids were determined using NMR and MS analyses, and in one instance, confirmed by X-ray diffraction analysis. The nor-6,7-secovallesamine alkaloid, pneumatophorine, is notable for an unusual incorporation of a 3-ethylpyridine moiety in a monoterpenoid indole. The rhazinilam-type alkaloids (rhazinicine, nor-rhazinicine, rhazinal, and rhazinilam) showed strong cytotoxicity toward human KB, HCT-116, MDA-MB-231, and MRC-5 cells, while pneumatophorine, the uleine alkaloid undulifoline, and the strychnan alkaloids, N4-demethylalstogustine and echitamidine, induced concentration dependent relaxation in phenylephrine-precontracted rat aortic rings.
Keywords: Alstonia pneumatophora; Alstonia rostrata; Apocynaceae; Indole alkaloids; NMR; Cytotoxicity; Vasorelaxation;

Anti-inflammation furanoditerpenoids from Caesalpinia minax Hance by Ruijuan Dong; Jiuzhi Yuan; Shilong Wu; Jian Huang; Xiaotong Xu; Zhaohua Wu; Huiyuan Gao (325-331).
Twelve furanocassane derivatives, including six previously unreported were isolated from the seeds of Caesalpinia minax Hance. Neocaesalminin A (1) was found with an unusual A-seco-rearranged cassane skeleton. The inhibitory effect on NO production for all compounds was evaluated in vitro. Furanoditerpenes were responsible for this herb’s anti-inflammation activity.Display OmittedCassane skeletons are rare in nature, but often possess valuable medicinal properties. A furanoditerpenoid with an unusual A-seco-rearranged cassane skeleton, neocaesalminin A, and five furanoditerpenoids were isolated from seeds of Caesalpinia minax Hance, along with six known cassane derivatives, 7-O-acetyl-bonducellpin C, caesalmin F, caesalmin C, ζ-caesalmin, caesalmin E1 and caesalpinin K. Compound structures were determined by spectroscopy (HR-ESI-MS, UV, IR, 1D NMR, 2D NMR), X-ray crystallography and quantum chemical computation of electronic circular dichroism). Three of the previously known compounds exhibited significant inhibition of nitric oxide production of RAW264.7 macrophages stimulated by lipopolysaccharide (LPS).
Keywords: Caesalpinia minax Hance; Caesalpinia; A-seco-rearranged cassane; Anti-inflammation activity; Circular dichroism; Conformation analysis;

The sesquiterpene lactone polymatin B from Smallanthus sonchifolius induces different cell death mechanisms in three cancer cell lines by Christian De Ford; Jerónimo L. Ulloa; César A.N. Catalán; Alfredo Grau; Virginia S. Martino; Liliana V. Muschietti; Irmgard Merfort (332-339).
The sesquiterpene lactone polymatin B induces different mechanisms of cell death which is dependent on the cell type. Preferentially, cell death is promoted by inducing oxidative stress in cancer cells.Display OmittedA 8β-angeloyloxy-9α-hydroxy-14-oxo-acanthospermolide and five known melampolide sesquiterpene lactones (uvedalin, enhydrin, polymatin B, sonchifolin, and fluctuanin) were isolated from the leaves of Smallanthus sonchifolius. The compounds were identified by 1D-, 2D-NMR, HRMS, IR and UV analyses. In vitro cytotoxicity assays (MTT) showed that these sesquiterpene lactones display poor cytotoxic effects on peripheral blood mononuclear cells (PBMC) of healthy human subjects, whereas a strong cytotoxicity was observed in leukemia and pancreas cancer cells. For the mechanism of action of polymatin B, oxidative stress seems to be involved. Interestingly, reactive oxygen species (ROS) formation mainly induced different effects: apoptosis in CCRF-CEM cells, necroptosis in CEM-ADR5000 cells through induction of RIP1K, neither apoptosis nor necroptosis in MIA-PaCa-2 cells. Additionally, cells also died partly by necrosis.
Keywords: Smallanthus sonchifolius; Asteraceae; Sesquiterpene lactones; Cytotoxicity;

Matairesinol of 10 and 100 μM extended the life span of C. elegans by 14% and 25% repectively.Display Omitted Arctium lappa is a well-known traditional medicinal plant in China (TCM) and Europe that has been used for thousands of years to treat arthritis, baldness or cancer. The plant produces lignans as secondary metabolites, which have a wide range of bioactivities. Yet, their antiaging potential has not been explored. In this study, we isolated six lignans from A. lappa seeds, namely arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C, and lappaol F. The antioxidant and antiaging properties of the isolated lignans were studied using Caenorhabditis elegans as a relevant animal model. All lignans at concentrations of 10 and 100 μM significantly extended the mean life span of C. elegans. The strongest effect was observed with matairesinol, which at a concentration of 100 μM extended the life span of worms by 25%. Additionally, we observed that five lignans are strong free radical-scavengers in vitro and in vivo and all lignans can improve survival of C. elegans under oxidative stress. Furthermore, the lignans can induce the nuclear translocation of the transcription factor DAF-16 and up-regulate its expression, suggesting that a possible underlying mechanism of the observed longevity-promoting activity of lignans depends on DAF-16 mediated signaling pathway. All lignans up-regulated the expression of jnk-1, indicating that lignans may promote the C. elegans longevity and stress resistance through a JNK-1-DAF-16 cascade. Our study reports new antiaging activities of lignans, which might be candidates for developing antiaging agents.
Keywords: Arctium lappa; Lignans; Caenorhabditis elegans; Antiaging agent; Free radical scavenger; DAF-16;

Alkyl hydroxycinnamates were found to be present in the root waxes of 10 of 11 species analyzed. Reduced forms of the photo-oxidation products of ω-hydroxy oleate and its corresponding dicarboxylate were identified as naturally occurring suberin monomers in rutabaga (Brassica napus subsp. rapifera) periderm tissues.Display OmittedAliphatic waxes can be found in association with suberized tissues, including roots. Non-polar lipids were isolated by rapid solvent extraction of mature regions of intact roots from eleven angiosperms, including both monocots and dicots. The majority of roots analyzed were taproots or tuberous taproots that had undergone secondary growth and thus were covered by a suberized periderm. The exceptions therein were maize (Zea mays L.) and rice (Oryza sativa L.), which present a suberized exodermis. The analysis herein focused on aliphatic waxes, with particular emphasis on alkyl hydroxycinnamates (AHCs). AHCs were widely distributed, absent from only one species, were found in both aerial and subterranean portions of tuberous taproots, and were associated with the fibrous roots of both maize and rice. Most species also contained monoacylglycerols, fatty alcohols and/or free fatty acids. Carrot (Daucus carrota L.) was the outlier, containing only free fatty acids, sterols, and polyacetylenes as identified components. Sterols were the only ubiquitous component across all roots analyzed. Monoacylglycerols of ω-hydroxy fatty acids were present in maize and rice root waxes. For species within the Brassiceae, wax compositions varied between subspecies or varieties and between aerial and subterranean portions of taproots. In addition, reduced forms of photo-oxidation products of ω-hydroxy oleate and its corresponding dicarboxylic acid (10,18-dihydroxy-octadec-8-enoate, 9,18-dihydroxy-octadec-10-enoate and 9-hydroxyoctadec-10-ene-1,18-dioate) were identified as naturally occurring suberin monomers in rutabaga (Brassica napus subsp. rapifera Metzg.) periderm tissues.
Keywords: Alkyl hydroxycinnamate; Suberin; Root wax; Suberin-associated waxes; Periderm; Exodermis; Photo-oxidation;

Molecular and biochemical characterization of the UDP-glucose: Anthocyanin 5-O-glucosyltransferase from Vitis amurensis by Fei He; Wei-Kai Chen; Ke-Ji Yu; Xiang-Nan Ji; Chang-Qing Duan; Malcolm J. Reeves; Jun Wang (363-372).
UDP-glucose: anthocyanin 5-O-glucosyltransferase from Vitis amurensis grapes (Va5GT), which catalyzes the glucosylation of anthocyanidin monoglucosides at the 5-O-position, is crucial for the biosynthesis of anthocyanidin bis-glucosides.Display OmittedGenerally, red Vitis vinifera grapes only contain monoglucosidic anthocyanins, whereas most non-vinifera red grapes of the Vitis genus have both monoglucosidic and bis-glucosidic anthocyanins, the latter of which are believed to be more hydrophilic and more stable. Although previous studies have established the biosynthetic mechanism for formation of monoglucosidic anthocyanins, less attention has been paid to that of bis-glucosidic anthocyanins. In the present research, the full-length cDNA of UDP-glucose: anthocyanin 5-O-glucosyltransferase from Vitis amurensis Rupr. cv. ‘Zuoshanyi’ grape (Va5GT) was cloned. After acquisition and purification of recombinant Va5GT, its enzymatic parameters were systematically analyzed in vitro. Recombinant Va5GT used malvidin-3-O-glucoside as its optimum glycosidic acceptor when UDP-glucose was used as the glycosidic donor. Va5GT-GFP was found to be located in the cytoplasm by analyzing its subcellular localization with a laser-scanning confocal fluorescence microscope, and this result was coincident with its metabolic function of modifying anthocyanins in grape cells. Furthermore, the relationship between the transcriptional expression of Va5GT and the accumulation of anthocyanidin bis-glucosides during berry development suggested that Va5GT is a key enzyme in the biosynthesis of bis-glucosidic anthocyanins in V. amurensis grape berries.
Keywords: Amur grape; Vitis amurensis; Vitaceae; Bis-glucosidic anthocyanin; UDP-glucose: anthocyanin 5-O-glucosyltransferase; Enzymology; Subcellular localization;

Abietic acid, dehydroabietic acid and squalene, isolated from Abies balsamea reduce glucose production and enhance glucose storage in liver cells.Display OmittedIn our previous study, Abies balsamea (L.) Mill., a plant used in Cree traditional medicine, had a strong effect on the regulation of glucose homeostasis in liver cells. This study aimed to isolate and identify its active constituents using a bioassay-guided fractionation approach as well as to elucidate their mechanism(s) of action. The effect of the crude extract and its constituents was evaluated on the activity of Glucose-6-Phosphatase (G6Pase) and Glycogen Synthase (GS) and phosphorylation of three kinases, AMP-activated protein kinase (AMPK), Akt and Glycogen Synthase Kinase-3 (GSK-3). Three compounds, abietic acid, dehydroabietic acid and squalene, were isolated from the most active fraction in the bioassays (hexane). The compounds were able to decrease the activity of G6Pase and to stimulate GS. Their effect on G6Pase activity involved both Akt and AMPK phosphorylation with significant correlations between insulin-dependent and -independent pathways and the bioassay. In addition, the compounds were able to stimulate GS through GSK-3 phosphorylation with a significant correlation between the signaling pathway and the bioassay. Dehydroabietic acid stood out for its strongest effect in all the experiments close to that of the crude extract. These compounds may have potential applications in the treatment of type 2 diabetes and insulin resistance.
Keywords: Balsam fir; Abies balsamea; Pinaceae; Type 2 diabetes; Traditional Aboriginal Medicine; Natural Health Product; Glucose-6-Phosphatase; Glycogen Synthase; Akt; AMPK; GSK-3;

Isolation and identification of Desmodium root exudates from drought tolerant species used as intercrops against Striga hermonthica by A.M. Hooper; J.C. Caulfield; B. Hao; J.A. Pickett; C.A.O. Midega; Z.R. Khan (380-387).
Drought tolerant Desmodium species, when mature, produce root exudates that inhibit Striga parasitism and which comprise a range of di-C-glycosylflavones.Display OmittedPlants from the genus Desmodium, in particular D. uncinatum, are used on sub-Saharan small-holder farms as intercrops to inhibit parasitism of cereal crops by Striga hermonthica and Striga asiatica via an allelopathic mechanism. The search for Desmodium species which are adapted to more arid conditions, and which show resilience to increased drought stress, previously identified D. intortum, D. incanum and D. ramosissimum as potential drought tolerant intercrops. Their potential as intercrops was assessed for resource poor areas of rain-fed cereal production where drought conditions can persist through normal meteorological activity, or where drought may have increasing impact through climate change. The chemical composition of the root exudates were characterised and the whole exudate biological activity was shown to be active in pot experiments for inhibition of Striga parasitism on maize. Furthermore, rain fed plot experiments showed the drought tolerant Desmodium intercrops to be effective for Striga inhibition. This work demonstrates the allelopathic nature of the new drought tolerant intercrops through activity of root exudates and the major compounds seen in the exudates are characterised as being C-glycosylflavonoid. In young plants, the exudates show large qualitative differences but as the plants mature, there is a high degree of convergence of the C-glycosylflavonoid exudate chemical profile amongst active Desmodium intercrops that confers biological activity. This defines the material for examining the mechanism for Striga inhibition.
Keywords: Desmodium uncinatum; Desmodium intortum; Desmodium incanum; Desmodium ramosissimum; Striga hermonthica; Parasitic weeds; Root exudate; Di-C-glycosylflavone; Striga inhibition;

Sequential oxidation of Jasmonoyl-Phenylalanine and Jasmonoyl-Isoleucine by multiple cytochrome P450 of the CYP94 family through newly identified aldehyde intermediates by Emilie Widemann; Bernard Grausem; Hugues Renault; Emmanuelle Pineau; Clément Heinrich; Raphaël Lugan; Pascaline Ullmann; Laurence Miesch; Yann Aubert; Michel Miesch; Thierry Heitz; Franck Pinot (388-399).
Members of the CYP94 family catalyze the production of oxidized derivatives of Jasmonoyl-Phenyalanine which accumulates in Arabidopsis leaves infected by Botrytis cinerea.Display OmittedThe role and fate of Jasmonoyl-Phenylalanine (JA-Phe), an understudied conjugate in the jasmonate pathway remain to be unraveled. We addressed here the possibility of JA-Phe oxidative turnover by cytochrome P450s of the CYP94 family. Leaf wounding or fungal infection in Arabidopsis resulted in accumulation of JA-Phe, 12-hydroxyl (12OH-JA-Phe) and 12-carboxyl (12COOH-JA-Phe) derivatives, with patterns differing from those previously described for Jasmonoyl-Isoleucine. In vitro, yeast-expressed cytochromes P450 CYP94B1, CYP94B3 and CYP94C1 differentially oxidized JA-Phe to 12-hydroxyl, 12-aldehyde and 12-carboxyl derivatives. Furthermore, a new aldehyde jasmonate, 12CHO-JA-Ile was detected in wounded plants. Metabolic analysis of CYP94B3 and CYP94C1 loss- and gain-of-function plant lines showed that 12OH-JA-Phe was drastically reduced in cyp94b3 but not affected in cyp94c1, while single or double mutants lacking CYP94C1 accumulated less 12COOH-JA-Phe than WT plants. This, along with overexpressing lines, demonstrates that hydroxylation by CYP94B3 and carboxylation by CYP94C1 accounts for JA-Phe turnover in planta. Evolutionary study of the CYP94 family in the plant kingdom suggests conserved roles of its members in JA conjugate homeostasis and possibly in adaptative functions. Our work extends the range and complexity of JA-amino acid oxidation by multifunctional CYP94 enzymes in response to environmental cues.
Keywords: Cytochrome P450; Hormone turnover; Signaling; Stress; Oxidation; Metabolomics; Jasmonic acid;

Diterpenoids from aerial parts of Flickingeria fimbriata and their nuclear factor-kappaB inhibitory activities by Hang Li; Jing-jun Zhao; Jin-long Chen; Long-ping Zhu; Dong-mei Wang; Lin Jiang; De-po Yang; Zhi-min Zhao (400-409).
Rare 16-nor-ent-pimarane type diterpenoids (15), two 15,16-dinor-ent-pimarane type (67) and one ent-pimarane diterpenoid (11), together with eight known analogs were isolated from Flickingeria fimbriata.Display OmittedChemical investigation of the aerial parts of Flickingeria fimbriata (Bl.) Hawkes resulted in isolation of sixteen ent-pimarane diterpenoids, including five rare 16-nor-ent-pimarane diterpenoids, two 15,16-dinor-ent-pimarane diterpenoids and one ent-pimarane diterpenoid. Structures were mainly elucidated by extensive spectroscopic analysis, and their absolute configurations were unequivocally determined by the exciton chirality method, the modified Mosher’s method, the CD experiments (including Snatzke’s method) and chemical transformations, respectively. All the isolated compounds were screened for inhibitory effects on the nuclear factor-kappaB (NF-κB) in lipopolysaccharide (LPS) induced murine macrophage RAW264.7 cells, using a NF-κB-dependent luciferase reporter gene assay. Several of these compounds displayed comparable or even better activities than the positive control pyrrolidinedithiocarbamate (PDTC) (IC50  = 26.3 μM) with IC50 values in the range of 14.7–29.2 μM and structure–activity relationships are briefly proposed.
Keywords: Flickingeria fimbriata; Orchidaceae; Norditerpenoids; Absolute configuration; NF-κB;

Further C-15-acyl phragmalin derivatives from Chukrasia tabularis A. Juss. by Jun Luo; Hong-Jian Zhang; Olga Quasie; Si-Ming Shan; Yang-Mei Zhang; Ling-Yi Kong (410-416).
Sixteen phragmalins with three types of enolic acyl substituents at C-15 were isolated from the seeds of Chukrasia tabularis A. Juss. One of these (compound 1) had its structure determined by NMR and X-ray diffraction analysis, while the others were determined via NMR, HR-MS, and CD spectra.Display OmittedRelying on characteristic double UV absorptions (210 and 270 nm), sixteen phragmalins with three types of enolic acyl substituents at C-15 were isolated directly from EtOH extracts of the seeds of Chukrasia tabularis A. Juss. Eight of these compounds possessed a C-15-acetyl phragmalin skeleton, and the basic carbon skeleton and absolute configuration of one of these was determined by NMR and X-ray diffraction analysis, while the structures of the other phragmalins were determined via NMR, HR-MS, and CD spectra. Additionally, all of the isolates were tested for inhibition against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 macrophages and cytotoxicity in SMMC-7721, MCF-7 and U2OS tumor cells.
Keywords: Chukrasia tabularis A. Juss.; Meliaceae; C-15 enolic acyl phragmalin; UV-guided isolation; Absolute configuration;

Sulfated phenolic acids from Dasycladales siphonous green algae by Caroline Kurth; Matthew Welling; Georg Pohnert (417-423).
Novel sulfated metabolites of Dasycladales are introduced that modulate biofilm formation after desulfatation.Display OmittedSulfated aromatic acids play a central role as mediators of chemical interactions and physiological processes in marine algae and seagrass. Among others, Dasycladus vermicularis (Scopoli) Krasser 1898 uses a sulfated hydroxylated coumarin derivative as storage metabolite for a protein cross linker that can be activated upon mechanical disruption of the alga. We introduce a comprehensive monitoring technique for sulfated metabolites based on fragmentation patterns in liquid chromatography/mass spectrometry and applied it to Dasycladales. This allowed the identification of two new aromatic sulfate esters 4-(sulfooxy)phenylacetic acid and 4-(sulfooxy)benzoic acid. The two metabolites were synthesized to prove the mass spectrometry-based structure elucidation in co-injections. We show that both metabolites are transformed to the corresponding desulfated phenols by sulfatases of bacteria. In biofouling experiments with Escherichia coli and Vibrio natriegens the desulfated forms were more active than the sulfated ones. Sulfatation might thus represent a measure of detoxification that enables the algae to store inactive forms of metabolites that are activated by settling organisms and then act as defense.
Keywords: Dasycladales; Anti-fouling metabolites; Siphonous green algae; Sulfatase; Activated defense; Sulfated phenolic acids; Marine chemical ecology;

Chemical constituents derived from Artocarpus xanthocarpus as inhibitors of melanin biosynthesis by Yu-Jing Jin; Cha-Chi Lin; Tzy-Ming Lu; Jih-Heng Li; Ih-Sheng Chen; Yueh-Hsiung Kuo; Horng-Huey Ko (424-435).
Six previously unknown compounds and 18 known compounds from Artocarpus xanthocarpus were isolated and characterized spectroscopically. Their anti-melanogenesis activity were evaluated in B16F10 melanoma cells. The results indicated that some of these constituents may be a candidate for the depigmentation agents.Display OmittedTwenty-four compounds, including the previously unknown artoxanthocarpuone A, artoxanthocarpuone B, hydroxylakoochin A, methoxylakoochin A, epoxylakoochin A, and artoxanthol, were isolated and characterized spectroscopically. Among them, artoxanthol is stilbene oligomer presumably constructed in a 5,11,12-triphenyl hexahydrochrysene scaffold by a Diels–Alder type of reaction, for which a biosynthetic pathway is proposed. Artoxanthol, alboctalol, steppogenin, norartocarpetin, resveratrol, oxyresveratrol, and chlorophorin potently inhibited mushroom tyrosinase activity with IC50 values from 0.9 to 5.7 μM that were all far stronger than the positive controls. Artoxanthocarpuone A, artoxanthocarpuone B, methoxylakoochin A, lakoochin A, cudraflavone C, artonin A, resveratrol, and chlorophorin reduced tyrosinase activity and inhibited α-melanocyte-stimulating hormone-induced melanin production in B16F10 melanoma cells without affecting cell proliferation. Collectively, the results suggest that the constituents of Artocarpus xanthocarpus have potential to be used as depigmentation agents.
Keywords: Chinese herb; Artocarpus xanthocarpus; Moraceae; Oligomer of stilbene; Hexahydrochrysene; Tyrosinase; Melanin;

Antimicrobial dihydrobenzofurans and xanthenes from a foliar endophyte of Pinus strobus by Susan N. Richardson; Tienabe K. Nsiama; Allison K. Walker; David R. McMullin; J. David Miller (436-443).
The screening of foliar endophytes of Pinus strobus led to the identification of fungus representing a previously unknown genus and species within the family Massarinaceae. From one isolate, four dihydrobenzofurans (14) and two xanthenes (56) were discovered and characterized. All of these elicited antifungal activity against the fungus Microbotryum violaceum and the Gram-positive bacterium Bacillus subtilis.Display OmittedFoliar fungal endophytes of Pinus strobus (eastern white pine) were collected from different sites across south-eastern New Brunswick, Canada and screened for the production of bioactive metabolites. From one site, two fungal isolates representing a formerly unknown genus and species within the family Massarinaceae (Pleosporales, Dothideomycetes, Ascomycota) were resolved by phylogenetic analysis. These isolates produced crude organic extracts that were active against Microbotryum violaceum and Saccharomyces cerevisiae. From these strains, DAOM 242779 and 242780, four dihydrobenzofurans (14) and two xanthenes (56) were characterized. Structures were elucidated by HRMS, interpretation of NMR spectra and other spectroscopic techniques. All isolated metabolites displayed antimicrobial activity against the biotrophic fungal pathogen M. violaceum and Bacillus subtilis.
Keywords: Endophytic fungi; Massarinaceae; Pinus strobus; Fungal metabolite; Dihydrobenzofuran; Xanthene; Antimicrobial activity; Microbotryum violaceum;

Display OmittedCultivation of grapes in West Bank – Palestine is very old and a large number of grape varieties exist as a result of continuous domestication over thousands of years. This rich biodiversity has highly influenced the consumer behavior of local people, who consume both grape berries and leaves. However, studies that address the contents of health-promoting metabolites in leaves are scarce. Accordingly the aim of this study is to assess metabolite levels in leaves of two grape varieties that were collected from semiarid and temperate regions. Metabolic profiling was conducted using GC–MS and LC–MS. The obtained results show that abiotic stresses in the semiarid region led to clear changes in primary metabolites, in particular in amino acids, which exist at very high levels. By contrast, qualitative and genotype-dependent differences in secondary metabolites were observed, whereas abiotic stresses appear to have negligible effect on the content of these metabolites. The qualitative difference in the flavonol profiles between the two genotypes is most probably related to differential expression of specific genes, in particular flavonol 3-O-rhamnosyltransferase, flavonol-3-O-glycoside pentosyltransferases and flavonol-3-O- d -glucoside l -rhamnosyltransferase by ‘Beituni’ grape leaves, which led to much higher levels of flavonols with rutinoside, pentoside, and rhamnoside moieties with this genotype.
Keywords: Grape; Vitis vinifera L.; Vitaceae; Metabolome; Flavonols; Stress;

The broad spectrum 2-oxoglutarate oxygenase inhibitor N-oxalylglycine is present in rhubarb and spinach leaves by Khalid Al-Qahtani; Bushra Jabeen; Rok Sekirnik; Naheed Riaz; Timothy D.W. Claridge; Christopher J. Schofield; James S.O. McCullagh (456-461).
The synthetic broad-spectrum 2-oxoglutarate oxygenase inhibitor N-oxalylglycine (NOG) is found to be present as a natural product in Rheum rhabarbarum (rhubarb) and Spinach oleracea (spinach) leaves but not Escherchia coli or human embryonic kidney cells (HEK 293T).Display Omitted2-Oxoglutarate (2OG) and ferrous iron dependent oxygenases are involved in many biological processes in organisms ranging from humans (where some are therapeutic targets) to plants. These enzymes are of significant biomedicinal interest because of their roles in hypoxic signaling and epigenetic regulation. Synthetic N-oxalylglycine (NOG) has been identified as a broad-spectrum 2OG oxygenase inhibitor and is currently widely used in studies on the hypoxic response and chromatin modifications in animals. We report the identification of NOG as a natural product present in Rheum rhabarbarum (rhubarb) and Spinach oleracea (spinach) leaves; NOG was not observed in Escherchia coli or human embryonic kidney cells (HEK 293T). The finding presents the possibility that NOG plays a natural role in regulating gene expression by inhibiting 2OG dependent oxygenases. This has significance because tricarboxylic acid cycle (TCA) intermediate inhibition of 2OG dependent oxygenases has attracted major interest in cancer research.
Keywords: Rheum rhabarbarum (rhubarb); Spinach oleracea (spinach); Natural product; Amino acid; N-oxalylglycine; Oxygenases; Inhibitor;

Cytotoxic diterpenoids from Jatropha curcas cv. nigroviensrugosus CY Yang Roots by JieQing Liu; YuanFeng Yang; JianJun Xia; XuYang Li; ZhongRong Li; Lin Zhou; MingHua Qiu (462-468).
Twenty diterpenoids were isolated from Jatropha curcas cv. nigroviensrugosus, one of them, lathyranlactone is an unusual diterpenoid lactone possessing a 5/13/3 tricyclic skeleton. Two of the previously known diterpenoids showed more potent cytotoxic activity against the SMMC-772, MCF-7 and SW480 human tumor cell lines than the positive control (cisplatin).Display OmittedAn investigation of phytochemicals from the roots of Jatropha curcas cv. nigroviensrugosus resulted in the isolation of twenty diterpenoids, including lathyranlactone, an unusual diterpenoid lactone possessing a 5/13/3 tricyclic skeleton, jatrocurcasenones A–E and jatrophodiones B–E, as well as 10 known analogues. All isolates were evaluated for cytotoxicity against the HL-60, SMMC-772, A-549, MCF-7 and SW480 human tumor cell lines using the MTS viability assay. Four of the known analogues showed cytotoxic activity in these cell lines, with IC50 values ranging from 2.0 to 23.0 μM. Moreover, the assessment of their cytotoxic structure–activity relationships showed the epoxy ring between C-5 and C-6 and the hydroxyl group at C-2 were the key functionalities for cytotoxicity.
Keywords: Jatropha curcas; Euphorbiaceae; Diterpenoids; Cytotoxic activity;

Repellency of α-pinene against the house fly, Musca domestica by Aaron T. Haselton; Angela Acevedo; Jacob Kuruvilla; Eric Werner; Jaydon Kiernan; Preeti Dhar (469-475).
The terpenes (1S)-(−)-α-pinene 1 and (1R)-(+)-α-pinene 2 exhibited repellent properties against house flies and both compounds elicited electrophysiological responses from in situ antennal preparations.Display Omitted Musca domestica L. is a non-biting nuisance fly that is capable of transmitting a large variety of pathogens to humans and non-human animals. Natural compounds and their derivatives, which are often less toxic than entirely synthetic compounds, may be used as repellents against M. domestica as part of comprehensive pest control and disease mitigation programs. This work investigates the repellent properties of the natural compound α-pinene against M. domestica.Adult house flies of both sexes avoided the volatile plant-derived terpenes (1S)-(−)-α-pinene 1 and (1R)-(+)-α-pinene 2 in constant air flow laboratory conditions, with 1 exhibiting a stronger repellent effect. House flies also avoided tarsal contact with filter paper saturated with 1. Furthermore, both 1 and 2 are electrophysiologically active on in situ female house fly antennal preparations.These findings demonstrate that α-pinene exhibits natural baseline repellency against the house fly, elicits a specific physiological response in this fly, and that functional or structural modification of 1 in particular may yield novel fly repellents with desirable properties.
Keywords: Y-tube olfactometer; Electroantennogram; Terpenes; Musca domestica; (1S)-(−) and (1R)-(+)-α-pinene; Repellent;

An unusual phenylpropanoid, 4-methoxycinnamic acid, was used in 13C-labeled form as a substrate to demonstrate its incorporation as an intact unit during the biosynthesis of 4′-methoxyanigorufone, which is reported here for the first time as a natural product.Display Omitted In vitro root cultures of Anigozanthos preissii and Wachendorfia thyrsiflora (Haemodoraceae) are suitable biological systems for studying the biosynthesis of phenylphenalenones. Here we report how we used these root cultures to investigate precursor–product relationships between phenylpropanoids and phenylphenalenones whose phenyl rings share identical substitution patterns. Four phenylpropanoic acids, including ferulic acid and the unusual 4-methoxycinnamic acid, were used in 13C-labeled form as substrates to study their incorporation into phenylphenalenones. In addition to the previously reported 2-hydroxy-9-(4′-hydroxy-3′-methoxyphenyl)-1H-phenalen-1-one (trivial name musanolone F), 2-hydroxy-9-(4′-methoxyphenyl)-1H-phenalen-1-one (proposed trivial name 4′-methoxyanigorufone) was found as a biosynthetic product in A. preissii. The carbon skeleton of 4′-methoxycinnamic acid was biosynthetically incorporated as an intact unit including its 4′-O-methyl substituent at the lateral phenyl ring. 4′-Methoxyanigorufone is reported here for the first time as a natural product.
Keywords: Anigozanthos preissii; Wachendorfia thyrsiflora; Biosynthesis; Haemodoraceae; Labeling; Nuclear magnetic resonance; Phenylphenalenones; Phenylpropanoids;

Phytotoxins produced by Phoma chenopodiicola, a fungal pathogen of Chenopodium album by Marco Evidente; Alessio Cimmino; Maria Chiara Zonno; Marco Masi; Alexander Berestetskyi; Ernesto Santoro; Stefano Superchi; Maurizio Vurro; Antonio Evidente (482-488).
Chenopodolan D, chenisocoumarin and chenopodolin B (1, 2 and 3) were isolated as new furopyran isocumarin and ent-pimaradiene from the culture filtrate of Phoma chenopodiicola. 1 and 3 showed phytotoxicity on non-host weeds.Display OmittedTwo phytotoxins were isolated from the liquid culture of Phoma chenopodiicola, a fungal pathogen proposed for the biological control of Chenopodium album, a common worldwide weed of arable crops. The two phytotoxins appeared to be a new tetrasubstituted furopyran and a new ent-pimaradiene. From the same culture a new tetrasubstituted isocoumarin was also isolated. These compounds were characterized by using spectroscopic (essentially 1D and 2D NMR and HR ESI MS) and chemical methods as 3-(3-methoxy-2,6-dimethyl-7aH-furo[2,3-b]pyran-4-yl)-but-2-en-1-ol (chenopodolan D, 1) (1S,2S,3S,4S,5S,9R,10S,12S,13S)-1,3,12-triacetoxy-2,hydroxy-6-oxo-ent-pimara-7(8),15-dien-18-oic acid 2,18-lactone (chenopodolin B, 3), and, 4,5,7-trihydroxy-3-methyl-isochroman-1-one (chenisocoumarin, 2) The absolute configuration of chenisocoumarin was assigned by applying an advanced Mosher’s method through the derivatization of its secondary hydroxylated carbon C-4, while that of chenopodolan D by application of quantum mechanical calculations of chiroptical (ECD and ORD) properties. When assayed by leaf puncture against non-host weeds, chenopodolan D and chenopodolin B showed phytotoxicity while chenisocoumarin and the 9-O-acetyl derivative of chenopodolan D were inactive. These results confirm that the nature of the side chain at C-4 in chenopodolans, and in particular its hydroxylation, are important features for activity. The activity of chenopodolin B could also be explained by its possible hydrolysis to chenopodolin.
Keywords: Chenopodium album; Phoma chenopodiicola; Phytotoxins; Furopyrans; ent-Pimaradiene; Isocoumarines; Herbicides;

The most appropriate method for the estimation of chlorogenic acids and their native derivatives in plants is SSDM because it does not induce any transformation and/or degradation processes in the analyzed substances.Display OmittedThe analytical procedures for determining plant constituents involve the application of sample preparation methods to fully isolate and/or pre-concentrate the analyzed substances. High-temperature liquid extraction is still applied most frequently for this purpose. The present paper shows that high-temperature extraction cannot be applied for the analysis of chlorogenic acids (CQAs) and their derivatives in plants as it causes the CQAs transformation leading to erroneous quantitative estimations of these compounds.Experiments performed on different plants (black elder, hawthorn, nettle, yerba maté, St John’s wort and green coffee) demonstrate that the most appropriate method for the estimation of CQAs/CQAs derivatives is sea sand disruption method (SSDM) because it does not induce any transformation and/or degradation processes in the analyzed substances. Owing to the SSDM method application we found that the investigated plants, besides four main CQAs, contain sixteen CQAs derivatives, among them three quinic acids.The application of SSDM in plant analysis not only allows to establish a true concentration of individual CQAs in the examined plants but also to determine which chlorogenic acids derivatives are native plant components and what is their concentration level. What is even more important, the application of SSDM in plant analysis allows to eliminate errors that may arise or might have arisen in the study of chlorogenic acids and their derivatives in plant metabolism.
Keywords: Sea sand disruption method (SSDM); Sample preparation; Plants analysis; Chlorogenic acids (CQAs); Chlorogenic acids derivatives;

Biotransformation of oleanolic and maslinic methyl esters by Rhizomucor miehei CECT 2749 by Antonio Martinez; Alberto Perojil; Francisco Rivas; Andres Parra; Andres Garcia-Granados; Antonia Fernandez-Vivas (500-508).
The biotransformation of methyl oleanolate (3), methyl maslinate (4), a 9(11),12-diene derivative (6) of methyl oleanolate, and a 12β,13β-epoxy derivative (13) of methyl maslinate, by Rhizomucor miehei CECT 2749, are described. Metabolites with hydroxylations at C-30, C-7β, or C-15α have been isolated. This fungus has also produced 9(11)-ene, 11-oxo, 12-oxo-13R, or 12-oxo-28,13β-olide derivatives.Display OmittedThe pentacyclic triterpenoids methyl oleanolate, methyl maslinate, methyl 3β-hydroxyolean-9(11),12-dien-28-oate, and methyl 2α,3β-dihydroxy-12β,13β-epoxyolean-28-oate were biotransformed by Rhizomucor miehei CECT 2749. Microbial transformation of methyl oleanolate produced only a 7β,30-dihydroxylated metabolite with a conjugated 9(11),12-diene system in the C ring. Biotransformation of the substrate with this 9(11),12-diene system gave the same 7β,30-dihydroxylated compound together with a 7β,15α,30-trihydroxyl derivative. The action of this fungus (R. miehei) on methyl maslinate was more varied, isolating metabolites with a 30-hydroxyl group, a 9(11),12-diene system, an 11-oxo group, or an 12-oxo group. Microbial transformation of the substrate with a 12β,13β-epoxy function resulted in the isolation of two metabolites with 12-oxo and 28,13β-olide groups, hydroxylated or not at C-7β, together with a 30-hydroxy-12-oxo derivative. The structures of these derivatives were deduced by extensive and rigorous spectroscopic studies.
Keywords: Rhizomucor miehei; Biotransformation; Biohydroxylation; Triterpenoids; Oleanolic acid; Maslinic acid; Methyl oleanolate; Methyl maslinate;

Molecular and biochemical characterization of mannitol-1-phosphate dehydrogenase from the model brown alga Ectocarpus sp. by Patricia Bonin; Agnès Groisillier; Alice Raimbault; Anaïs Guibert; Catherine Boyen; Thierry Tonon (509-520).
Mannitol metabolism plays key roles in the physiology of brown algae. This article describes the biochemical characterization of the recombinant catalytic domain of one mannitol-1-phosphate dehydrogenase (M1PDH) of the model organism Ectocarpus sp.Display OmittedThe sugar alcohol mannitol is important in the food, pharmaceutical, medical and chemical industries. It is one of the most commonly occurring polyols in nature, with the exception of Archaea and animals. It has a range of physiological roles, including as carbon storage, compatible solute, and osmolyte. Mannitol is present in large amounts in brown algae, where its synthesis involved two steps: a mannitol-1-phosphate dehydrogenase (M1PDH) catalyzes a reversible reaction between fructose-6-phosphate (F6P) and mannitol-1-phosphate (M1P) (EC 1.1.1.17), and a mannitol-1-phosphatase hydrolyzes M1P to mannitol (EC 3.1.3.22). Analysis of the model brown alga Ectocarpus sp. genome provided three candidate genes for M1PDH activities. We report here the sequence analysis of Ectocarpus M1PDHs (EsM1PDHs), and the biochemical characterization of the recombinant catalytic domain of EsM1PDH1 (EsM1PDH1cat). Ectocarpus M1PDHs are representatives of a new type of modular M1PDHs among the polyol-specific long-chain dehydrogenases/reductases (PSLDRs). The N-terminal domain of EsM1PDH1 was not necessary for enzymatic activity. Determination of kinetic parameters indicated that EsM1PDH1cat displayed higher catalytic efficiency for F6P reduction compared to M1P oxidation. Both activities were influenced by NaCl concentration and inhibited by the thioreactive compound pHMB. These observations were completed by measurement of endogenous M1PDH activity and of EsM1PDH gene expression during one diurnal cycle. No significant changes in enzyme activity were monitored between day and night, although transcription of two out of three genes was altered, suggesting different levels of regulation for this key metabolic pathway in brown algal physiology.
Keywords: Brown algae; Ectocarpus sp.; Mannitol cycle; Mannitol-1-phosphate dehydrogenase; Recombinant protein;

Antrocarines A–F, antiplasmodial ergostane steroids from the stem bark of Antrocaryon klaineanum by Pascal D. Douanla; Turibio Kuiate Tabopda; Alembert T. Tchinda; Ewa Cieckiewicz; Michel Frédérich; Fabrice Fekam Boyom; Nole Tsabang; Samuel Yeboah; Augustin E. Nkengfack; Marguerite Hortence K. Tchuendem (521-526).
Six new ergostane derivatives, together with two known compounds, were isolated from Antrocaryon klaineanum. The compounds were evaluated for their ability to inhibit the 3D7 strain of Plasmodium falciparum. Compounds 2, 3, and 8 showed potent activity.Display OmittedDuring a study on the chemistry and biological activity of Antrocaryon klaineanum Pierre, six new sterols including 4,24(28)-ergostadiene-6α,7α-diol (1), 6α-methoxy-4,24(28)-ergostadiene-7α,20S-diol (2), 6α-methoxy-4,24(28)-ergostadien-7α-ol (3), 20S-hydroxy-24(28)-ergosten-3-one (4), 7α-hydroxy-4,24(28)-ergostadien-3-one (5), and 24(28)-ergostene-3β,6α-diol (6) were characterized by physical and spectroscopic means. The known steroids 7 and 8 were also isolated. The crude extract and the isolated compounds were evaluated for their ability to inhibit the 3D7 strain of Plasmodium falciparum. Compounds 2, 3, and 8 showed potent activity while that of the crude extract was moderate.
Keywords: Antrocaryon klaineanum; Anacardiaceae; Ergostane steroids; Antiplasmodial activities;

Exogenous plant hormones and cyclotide expression in Viola uliginosa (Violaceae) by Blazej Slazak; Erik Jacobsson; Elżbieta Kuta; Ulf Göransson (527-536).
The regulation of cyclotide expression is related to plant hormones governing tissue development. The production of cyclotides can be controlled in vitro.Display OmittedPlants from Violaceae produce cyclotides, peptides characterized by a circular peptide backbone and a cystine knot. This signature motif gives stability that can harness a wide spectrum of biological activities, with implications in plant defense and with applications in medicine and biotechnology. In the current work, cyclotide expressing in vitro cultures were established from Viola uliginosa. These cultures are useful models for studying biosynthesis of cyclotides and can also be used in their production. The cyclotide expression pattern is shown to be dependent on exogenous plant growth regulators, both on peptide and gene expression levels. The highest yields of cyclotides were obtained on media containing only a cytokinin and were correlated with storage material accumulation. Exposure to auxins decreased cyclotide production and caused shifting of the biosynthesis pattern to root specific cyclotides. The response to stimuli in terms of cyclotide expression pattern appears to be developmental, and related to polar auxin transportation and the auxin/cytokinin ratio regulating tissue differentiation. By the use of whole transcriptome shotgun sequencing (WTSS) and peptidomics, 20 cyclotide sequences from V. uliginosa (including 12 new) and 12 complete precursor proteins could be identified. The most abundant cyclotides were cycloviolacin O3 (CyO3), CyO8 and CyO13. A suspension culture was obtained that grew exponentially with a doubling time of approximately 3 days. After ten days of growth, the culture provided a yield of more than 4 mg CyO13 per gram dry mass.
Keywords: Viola uliginosa (Violaceae); Cyclotides; In vitro culture; Plant growth regulators; Whole transcriptome shotgun sequencing; Mass spectrometry;

The co-expression of vacuolar NHX and proton pump from wheat imparted salinity tolerance in tomato. Transgenic tomato fruits accumulate more K+ than their WT counterpart.Display OmittedPotassium (K) deficiency is a worldwide problem. Thus, the K biofortification of crops is needed to enhance human nutrition. Tomato represents an ideal candidate for such biofortification programs thanks to its widespread distribution and its easy growth on a commercial scale. However, although tomato is moderately tolerant to abiotic stresses, the crop losses due to salinity can be severe. In this study, we generated transgenic tomato plants over-expressing a Na+–K+/H+ exchanger gene (TNHXS1), singly or with H+-pyrophosphatase (H+-PPiase) gene using a bicistronic construct. Transgenic tomato lines co-expressing both genes (LNV) significantly showed higher salinity tolerance than the wild-type (WT) plans or those expressing the TNHXS1 gene alone (LN). Indeed, under salt stress conditions, double transgenic plants produced higher biomass and retained more chlorophyll and catalase (CAT) activity. In addition, they showed earlier flowering and produced more fruits. To address K deficiencies in humans, an increase of 50% in K content of vegetable products was proposed. In this study, ion content analysis revealed that, under salt stress, fruits from double transgenic plants accumulated 5 times more potassium and 9 times less sodium than WT counterparts. Interestingly, the ionomic analysis of tomato fruits also revealed that LNV had a distinct profile compared to WT and to LN plants. Indeed, LNV fruits accumulated less Fe2+, Ca2+, Mg2+ and Zn2+, but more Mn2+. This study demonstrates the effectiveness of bicistronic constructs as an important tool for the enhancement of biofortification and salt stress tolerance in crops.
Keywords: Tomato; Biofortification; NHX-type protein; Pyrophosphatase; Potassium; Salt tolerance; IRES; Dicistronic vector; Ionomic analysis;

Abscisic acid induces biosynthesis of bisbibenzyls and tolerance to UV-C in the liverwort Marchantia polymorpha by Akito Kageyama; Kimitsune Ishizaki; Takayuki Kohchi; Hideyuki Matsuura; Kosaku Takahashi (547-553).
ABA and UV-C induced the biosynthesis of bisbibenzyls in the model liverwort Marchantia polymorpha, and UV-C irradiation was severely damaging to the MpABI1 overexpressor.Display OmittedEnvironmental stresses are effective triggers for the biosynthesis of various secondary metabolites in plants, and phytohormones such as jasmonic acid and abscisic acid are known to mediate such responses in flowering plants. However, the detailed mechanism underlying the regulation of secondary metabolism in bryophytes remains unclear. In this study, the induction mechanism of secondary metabolites in the model liverwort Marchantia polymorpha was investigated. Abscisic acid (ABA) and ultraviolet irradiation (UV-C) were found to induce the biosynthesis of isoriccardin C, marchantin C, and riccardin F, which are categorized as bisbibenzyls, characteristic metabolites of liverworts. UV-C led to the significant accumulation of ABA. Overexpression of MpABI1, which encodes protein phosphatase 2C (PP2C) as a negative regulator of ABA signaling, suppressed accumulation of bisbibenzyls in response to ABA and UV-C irradiation and conferred susceptibility to UV-C irradiation. These data show that ABA plays a significant role in the induction of bisbibenzyl biosynthesis, which might confer tolerance against UV-C irradiation in M. polymorpha.
Keywords: Common liverwort; Marchantiophyta; Marchantia polymorpha; Abscisic acid; Bisbibenzyl; Secondary metabolite; UV-C irradiation;

Gingerols and shogaols: Important nutraceutical principles from ginger by Ruchi Badoni Semwal; Deepak Kumar Semwal; Sandra Combrinck; Alvaro M. Viljoen (554-568).
Gingerols and the corresponding shogaols are the major pungent compounds present in the rhizomes of fresh and dried ginger (Zingiber officinale), respectively. The compounds have a favourable toxicity profile, but are cytotoxic towards a range of cancer cell lines. Tumours induced in several animal models were successfully treated by gingerols.Display OmittedGingerols are the major pungent compounds present in the rhizomes of ginger (Zingiber officinale Roscoe) and are renowned for their contribution to human health and nutrition. Medicinal properties of ginger, including the alleviation of nausea, arthritis and pain, have been associated with the gingerols. Gingerol analogues are thermally labile and easily undergo dehydration reactions to form the corresponding shogaols, which impart the characteristic pungent taste to dried ginger. Both gingerols and shogaols exhibit a host of biological activities, ranging from anticancer, anti-oxidant, antimicrobial, anti-inflammatory and anti-allergic to various central nervous system activities. Shogaols are important biomarkers used for the quality control of many ginger-containing products, due to their diverse biological activities. In this review, a large body of available knowledge on the biosynthesis, chemical synthesis and pharmacological activities, as well as on the structure–activity relationships of various gingerols and shogaols, have been collated, coherently summarised and discussed. The manuscript highlights convincing evidence indicating that these phenolic compounds could serve as important lead molecules for the development of therapeutic agents to treat various life-threatening human diseases, particularly cancer. Inclusion of ginger or ginger extracts in nutraceutical formulations could provide valuable protection against diabetes, cardiac and hepatic disorders.
Keywords: Zingiber officinale; Ginger; Nutraceutical; Gingerols; Shogaols; Antidiabetic; Anticancer; Anti-inflammatory;