Phytochemistry (v.111, #C)

Noscapine comes of age by Xue Chen; Thu-Thuy T. Dang; Peter J. Facchini (7-13).
Noscapine is a major alkaloid in opium poppy that has been used as a cough suppressant and investigated as a potential anticancer drug. A history of important phytochemical and pharmacological discoveries, including recent progress toward elucidation of the noscapine biosynthetic pathway, is presented.Display OmittedNoscapine is a phthalideisoquinoline alkaloid, which represents a class of plant specialized metabolites within the large and structurally diverse group of benzylisoquinoline alkaloids. Along with the narcotic analgesic morphine, noscapine is a major alkaloid in the latex of opium poppy (Papaver somniferum) that has long been used as a cough suppressant and has undergone extensive investigation as a potential anticancer drug. Cultivated opium poppy plants remain the only commercial source of noscapine. Despite its isolation from opium more than two centuries ago, the almost complete biosynthesis of noscapine has only recently been established based on an impressive combination of molecular genetics, functional genomics, and metabolic biochemistry. In this review, we provide a historical account of noscapine from its discovery through to initial investigations of its formation in opium poppy. We also describe recent breakthroughs that have led to an elucidation of the noscapine biosynthetic pathway, and we discuss the pharmacological properties that have prompted intensive evaluation of the potential pharmaceutical applications of noscapine and several semi-synthetic derivatives. Finally, we speculate on the future potential for the production of noscapine using metabolic engineering and synthetic biology in plants and microbes.
Keywords: Anticancer drugs; Benzylisoquinoline alkaloids; Cough suppressant; Noscapine; Phthalideisoquinoline alkaloids; Specialized metabolism;

Allocate carbon for a reason: Priorities are reflected in the 13C/12C ratios of plant lipids synthesized via three independent biosynthetic pathways by Youping Zhou; Hilary Stuart-Williams; Kliti Grice; Zachary E. Kayler; Saša Zavadlav; Angela Vogts; Florian Rommerskirchen; Graham D. Farquhar; Arthur Gessler (14-20).
The pathway-dependent 13C/12C patterns of higher plant lipids can be adequately explained by lipid pathway carbon allocation priorities.Display OmittedIt has long been theorized that carbon allocation, in addition to the carbon source and to kinetic isotopic effects associated with a particular lipid biosynthetic pathway, plays an important role in shaping the carbon isotopic composition (13C/12C) of lipids (Park and Epstein, 1961). If the latter two factors are properly constrained, valuable information about carbon allocation during lipid biosynthesis can be obtained from carbon isotope measurements. Published work of Chikaraishi et al. (2004) showed that leaf lipids isotopic shifts from bulk leaf tissue Δδ 13C bk lp (defined as δ 13Cbulkleaftissue  −  δ 13Clipid) are pathway dependent: the acetogenic (ACT) pathway synthesizing fatty lipids has the largest isotopic shift, the mevalonic acid (MVA) pathway synthesizing sterols the lowest and the phytol synthesizing 1-deoxy-d-xylulose 5-phosphate (DXP) pathway gives intermediate values. The differences in Δδ 13C bk lp between C3 and C4 plants Δ δ 13 C bk - lp , C 4 - C 3 are also pathway-dependent: Δ δ 13 C bk - lp , C 4 - C 3 ACT  >  Δ δ 13 C bk - lp , C 4 - C 3 DXP  >  Δ δ 13 C bk - lp , C 4 - C 3 MVA . These pathway-dependent differences have been interpreted as resulting from kinetic isotopic effect differences of key but unspecified biochemical reactions involved in lipids biosynthesis between C3 and C4 plants. After quantitatively considering isotopic shifts caused by (dark) respiration, export-of-carbon (to sink tissues) and photorespiration, we propose that the pathway-specific differences Δ δ 13 C bk - lp , C 3 - C 4 can be successfully explained by C4  − C3 carbon allocation (flux) differences with greatest flux into the ACT pathway and lowest into the MVA pathways (when flux is higher, isotopic shift relative to source is smaller). Highest carbon allocation to the ACT pathway appears to be tied to the most stringent role of water-loss-minimization by leaf waxes (composed mainly of fatty lipids) while the lowest carbon allocation to the MVA pathway can be largely explained by the fact that sterols act as regulatory hormones and membrane fluidity modulators in rather low concentrations.
Keywords: Natural abundance carbon isotopes; Lipid biosynthetic pathways; Isotope effects; Carbon allocation priority; C3  − C4 difference;

The moderate affinity of GL3 towards the inhibitor MYBL2, and higher affinity of GL3 for the positive regulator PAP2, facilitate the function of GL3 as an activator of anthocyanin synthesis.Display OmittedThe two Arabidopsis basic-helix-loop-helix transcription factors GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) are positive regulators of anthocyanin biosynthesis, and form protein complexes (MBW complexes) with various R2R3 MYB transcription factors and a WD40 repeat protein TRANSPARENT TESTA GLABROUS1 (TTG1). In earlier studies, GL3, in contrast to EGL3, was shown to be essential for accumulation of anthocyanins in response to nitrogen depletion. This could not be fully explained by the strong induction of GL3 in response to nitrogen depletion because the EGL3 transcripts were constitutively at a relatively high level and transcripts levels of the two genes were similar under nitrogen depletion. Here the GL3 and EGL3 proteins were characterized with respect to their affinities for PRODUCTION OF ANTHOCYANIN PIGMENT2 (PAP2), a R2R3-MYB which is induced by nitrogen depletion and is part of MBW complexes promoting anthocyanin synthesis. GL3 and EGL3 were also tested for their binding to MYBL2, a negative regulator of anthocyanin synthesis and MBW complexes. Using heterologously expressed proteins and Microscale Thermophoresis, GL3 showed binding constants (Kd ) of 3.5 ± 1.7 and 22.7 ± 3.7 μM, whereas EGL3 showed binding constants of 7.5 ± 2.3 and 8.9 ± 1.4 μM for PAP2 and MYBL2, respectively. This implies that MYBL2 will not inhibit a MBW complex containing GL3 as easily as for a complex containing EGL3. In transgenic plants where EGL3 reaches high concentrations compared with MYBL2 the equilibrium is shifted and MYBL2 is not likely to be an efficient competitor, hence anthocyanin formation could be restored by either EGL3 or GL3 genes when overexpressed by help of the 35S promoter. The present work underpins that GL3 is essential for anthocyanin accumulation under nitrogen depletion not only due to transcriptional activation, but also because of binding properties to proteins promoting or inhibiting the activity of the MBW complex.
Keywords: Arabidopsis thaliana; Anthocyanins; bHLH; EGL3; DFR; GL3; MYBL2; Nitrogen deficiency; PAP2;

Cloning, heterologous expression and biochemical characterization of plastidial sn-glycerol-3-phosphate acyltransferase from Helianthus annuus by Miriam Payá-Milans; Mónica Venegas-Calerón; Joaquín J. Salas; Rafael Garcés; Enrique Martínez-Force (27-36).
Biochemical characterization of recombinant GPAT showed an oleate specific isoform in sunflower plastids, concomitant with plant resistance to chilling temperatures.Display OmittedThe acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyzes the first step of glycerolipid assembly within the stroma of the chloroplast. In the present study, the sunflower (Helianthus annuus, L.) stromal GPAT was cloned, sequenced and characterized. We identified a single ORF of 1344 base pairs that encoded a GPAT sharing strong sequence homology with the plastidial GPAT from Arabidopsis thaliana (ATS1, At1g32200). Gene expression studies showed that the highest transcript levels occurred in green tissues in which chloroplasts are abundant. The corresponding mature protein was heterologously overexpressed in Escherichia coli for purification and biochemical characterization. In vitro assays using radiolabelled acyl-ACPs and glycerol-3-phosphate as substrates revealed a strong preference for oleic versus palmitic acid, and weak activity towards stearic acid. The positional fatty acid composition of relevant chloroplast phospholipids from sunflower leaves did not reflect the in vitro GPAT specificity, suggesting a more complex scenario with mixed substrates at different concentrations, competition with other acyl-ACP consuming enzymatic reactions, etc. In summary, this study has confirmed the affinity of this enzyme which would partly explain the resistance to cold temperatures observed in sunflower plants.
Keywords: Helianthus annuus; Sunflower; Substrate specificity; Glycerol-3-phosphate acyltransferase; Plastids;

Allosteric substrate inhibition of Arabidopsis NAD-dependent malic enzyme 1 is released by fumarate by Marcos Ariel Tronconi; Mariel Claudia Gerrard Wheeler; Andrea Martinatto; Juan Pablo Zubimendi; Carlos Santiago Andreo; María Fabiana Drincovich (37-47).
l-Malate binding to the allosteric site of NAD-ME1 elicits a sigmoidal and low substrate-affinity response (A). Fumarate binding turns NAD-ME1 into a hyperbolic and high substrate affinity enzyme (B).Display OmittedPlant mitochondria can use l-malate and fumarate, which accumulate in large levels, as respiratory substrates. In part, this property is due to the presence of NAD-dependent malic enzymes (NAD-ME) with particular biochemical characteristics. Arabidopsis NAD-ME1 exhibits a non-hyperbolic behavior for the substrate l-malate, and its activity is strongly stimulated by fumarate. Here, the possible structural connection between these properties was explored through mutagenesis, kinetics, and fluorescence studies. The results indicated that NAD-ME1 has a regulatory site for l-malate that can also bind fumarate. l-Malate binding to this site elicits a sigmoidal and low substrate-affinity response, whereas fumarate binding turns NAD-ME1 into a hyperbolic and high substrate affinity enzyme. This effect was also observed when the allosteric site was either removed or altered. Hence, fumarate is not really an activator, but suppresses the inhibitory effect of l-malate. In addition, residues Arg50, Arg80 and Arg84 showed different roles in organic acid binding. These residues form a triad, which is the basis of the homo and heterotrophic effects that characterize NAD-ME1. The binding of l-malate and fumarate at the same allosteric site is herein reported for a malic enzyme and clearly indicates an important role of NAD-ME1 in processes that control flow of C4 organic acids in Arabidopsis mitochondrial metabolism.
Keywords: Arabidopsis thaliana; Cruciferae; Regulatory site; Mitochondria; Malic enzyme; l-Malate; Fumarate;

Cloning and functional characterization of a 4-coumarate CoA ligase from liverwort Plagiochasma appendiculatum by Shuai Gao; Hai-Na Yu; Rui-Xue Xu; Ai-Xia Cheng; Hong-Xiang Lou (48-58).
Pa4CL1 showed high activity toward p-coumaric acid, along with the conversion of cinnamic acid and caffeic acid to their corresponding CoA thioesters; in particular, the enzyme participates in the conversion of dihydro-p-coumaric acid into dihydro-p-coumaroyl CoA, which is the precursor for bis-bibenzyl synthesis in liverworts. Display OmittedPlant phenylpropanoids represent a large group of secondary metabolites which have played an important role in terrestrial plant life, beginning with the evolution of land plants from primitive green algae. 4-Coumarate: coenzyme A ligase (4CL) is a provider of activated thioester substrates within the phenylpropanoid synthesis pathway. Although 4CLs have been extensively characterized in angiosperm, gymnosperm and moss species, little is known of their functions in liverworts. Here, a 4CL homolog (designated as Pa4CL1) was isolated from the liverwort species Plagiochasma appendiculatum. The full-length cDNA sequence of Pa4CL1 contains 1644 bp and is predicted to encode a protein with 547 amino acids. The gene products were 40–50% identical with 4CL sequences reported in public databases. The recombinant protein was heterologously expressed in Escherichia coli and exhibited a high level of 4CL activity, catalyzing formation of hydroxycinnamate-CoA thioesters by a two-step reaction mechanism from corresponding hydroxycinnamic acids. Kinetic analysis indicated that the most favorable substrate for Pa4CL1 is p-coumaric acid. The transcription of Pa4CL1 was induced when P. appendiculatum thallus was treated with either salicylic acid or methyl jasmonate.
Keywords: Liverwort; Plagiochasma appendiculatum; Marchantiales; Aytoniaceae; 4-Coumarate: coenzyme A ligase; Phenylpropanoids; Flavonoids; Bis-bibenzyls;

3β-Hydroxy-5β-cholestan-6-one was characterized as an early-step intermediate for 20-hydroxyecdysone biosynthesis in Ajuga hairy roots. 2H-labeled cholesterol was converted to the ketone with migration of the hydrogen atom from C-6 to C-5. The ketone was suggested to be an intermediate immediately after cholesterol in this plant.Display Omitted3β-Hydroxy-5β-cholestan-6-one was identified in the EtOAc extract of Ajuga hairy roots by micro-analysis using LC–MS/MS in the multiple reaction mode (MRM). Furthermore, administration of (2,2,4,4,7,7-2H6)- and (2,2,4,4,6,7,7-2H7)-cholesterols to the hairy roots followed by LC–MS/MS analysis of the EtOAc extract of the hairy roots indicated that cholesterol was converted to the 5β-ketone with hydrogen migration from the C-6 to the C-5 position. These findings, in conjunction with the previous observation that the ketone was efficiently converted to 20-hydroxyecdysone, strongly suggest that the 5β-ketone is an intermediate immediately formed after cholesterol during 20-hydroxyecdysone biosynthesis in Ajuga sp. In addition, the mechanism of the 5β-ketone formation from cholesterol is discussed.
Keywords: Hairy root; Ajuga reptans var. atropurpurea; Labiatae; 20-Hydroxyecdysone; Phytoecdysteroid; LC–MS/MS; NIH shift;

The effects of exogenous application of auxin (IAA) on oil content and fatty acid composition of C. vulgaris (UMT-M1) at early stationary growth phase.Display OmittedMicroalgae lipids and oils are potential candidates for renewable biodiesel. Many microalgae species accumulate a substantial amount of lipids and oils under environmental stresses. However, low growth rate under these adverse conditions account for the decrease in overall biomass productivity which directly influence the oil yield. This study was undertaken to investigate the effect of exogenously added auxin (indole-3-acetic acid; IAA) on the oil content, fatty acid compositions, and the expression of fatty acid biosynthetic genes in Chlorella vulgaris (UMT-M1). Auxin has been shown to regulate growth and metabolite production of several microalgae. Results showed that oil accumulation was highest on days after treatment (DAT)-2 with enriched levels of palmitic (C16:0) and stearic (C18:0) acids, while the linoleic (C18:2) and α-linolenic (C18:3n3) acids levels were markedly reduced by IAA. The elevated levels of saturated fatty acids (C16:0 and C18:0) were consistent with high expression of the β-ketoacyl ACP synthase I (KAS I) gene, while low expression of omega-6 fatty acid desaturase (ω-6 FAD) gene was consistent with low production of C18:2. However, the increment of stearoyl-ACP desaturase (SAD) gene expression upon IAA induction did not coincide with oleic acid (C18:1) production. The expression of omega-3 fatty acid desaturase (ω-3 FAD) gene showed a positive correlation with the synthesis of PUFA and C18:3n3.
Keywords: Microalgae; Chlorella vulgaris; Chlorellaceae; Indole-3-acetic acid; Keto-acyl ACP synthase I; Stearoyl-ACP desaturase; Omega-3 fatty acid desaturase; Omega-6 fatty acid desaturase;

Fruits from ripening impaired, chlorophyll degraded and jasmonate insensitive tomato mutants have altered tocopherol content and composition by Juliana Almeida; Ramón Asís; Virginia Noel Molineri; Ivan Sestari; Bruno Silvestre Lira; Fernando Carrari; Lázaro Eustáquio Pereira Peres; Magdalena Rossi (72-83).
An integrated analysis of metabolite and gene expression profiles dissects isoprenoid metabolism in ripening-impaired, senescence-related and jasmonate insensitive tomato mutants.Display OmittedSince isoprenoids are precursors in chlorophyll, carotenoid and tocopherol pathways, the study of their metabolism is of fundamental importance in understanding the regulatory cross-talk that contributes to the nutritional quality of tomato fruits. By means of an integrated analysis of metabolite and gene expression profiles, isoprenoid metabolism was dissected in ripening-impaired (ripening inhibitor and non-ripening), senescence-related (lutescent1 and green flesh) and jasmonate insensitive (jasmonic acid insensitive 1-1) tomato mutants, all in the Micro-Tom genetic background. It was found that the more upstream the location of the mutated gene, the more extensive the effect on the transcriptional profiles of the isoprenoid-related genes. Although there was a distinct effect in the analyzed mutations on chlorophyll, carotenoid and tocopherol metabolism, a metabolic adjustment was apparent such the antioxidant capacity mostly remained constant. Transcriptional profiles from fruits of ripening and senescence-related tomato mutants suggested that maintenance of the de novo phytyl diphosphate synthesis might, in later ripening stages, compensate for the lack of chlorophyll-derived phytol used in tocopherol production. Interestingly, an impairment in jasmonate perception led to higher total tocopherol levels in ripe fruits, accompanied by an increase in antioxidant capacity, highlighting the contribution of tocopherols to this nutritionally important trait.
Keywords: Tomato mutants; Solanum lycopersicum; Solanaceae; Atocopherol; Carotenoids; Chlorophyll; Jasmonic acid; Ripening; Antioxidant capacity;

Biosynthetic relationship between C28-brassinosteroids and C29-brassinosteroids in rice (Oryza sativa) seedlings by Se-Hwan Joo; Mun-Seok Jang; Min Kyun Kim; Ji-Eun Lee; Seong-Ki Kim (84-90).
28-Homocasterone is converted to castasterone by C-28 demethylation which indicates that biosynthesis of 28-homocastasterone is an alternative route to generate castasterone in rice.Display OmittedA crude enzyme solution was prepared from young rice seedlings, and the metabolism of C29-brassinosteroids identified from the seedlings was examined. When 28-homoteasterone was added as a substrate, 28-homotyphasterol, teasterone, and 26-nor-28-homoteasterone were characterized as enzyme products by GC–MS/SIM analysis. With 28-homotyphasterol, 28-homoteasterone, typhasterol, 28-homocastasterone, and 26-nor-28-homotyphasterol were formed and identified as products. When 28-homocastasterone was used, castasterone and 26-nor-28-homocastasterone were identified as products. Together with the reduced biological activity of C29-brassinosteroids and their metabolites in the rice lamina inclination assay, these metabolic studies suggest a biosynthetic sequence, 28-homoteasterone ↔ 28-homotyphasterol → 28-homocastasterone for C29-brassinosteroid biosynthesis is connected to the biosynthetic sequence teasterone ↔ typhasterol → castasterone for C28-brassinosteroids by C-28 demethylation, i.e., in order to increase biological activity in the rice plant. Additionally, the C29-brassinosteroids seem to bio-degrade their C-26 demethylated C28-brassinosteroid analogs to reduce brassinosteroid activity in planta. In conclusion, the biosynthesis of C29-brassinosteroids is a likely alternative route to the biologically-active brassinosteroid, castasterone, in rice.
Keywords: C29-brassinosteroids; Biosynthesis; C-28 demethylation; Castasterone; Rice; Oryza sativa; Gramineae;

The main strigolactone produced by the model legume Medicago truncatula was isolated and identified.Display OmittedA major strigolactone produced by the model legume Medicago truncatula (barrel medic) has been tentatively identified as a didehydro-orobanchol isomer. In this study, a putative didehydro-orobanchol isomer was isolated from root exudates collected from barrel medic grown hydroponically under phosphate-starved conditions. The structure and absolute configurations of this strigolactone, named medicaol, were determined by a combination of spectroscopic analysis and spectral comparison with 4-deoxymedicaol which was synthesized using solvolysis and rearrangement of hydroxymethylhexahydroindenone to tetrahydroazulenone as a key step. Medicaol has a seven-membered cycloheptadiene in the A ring instead of a typical six-membered cyclohexene. Medicaol and 4-deoxymedicaol showed activity comparable to their corresponding six-membered A ring relatives, orobanchol and 4-deoxyorobanchol (ent-2′-epi-5-deoxystrigol), in inducing hyphal branching of germinating spores of an arbuscular mycorrhizal fungus Gigaspora margarita. Plausible biosynthetic pathways from 4-deoxyorobanchol to medicaol are also proposed.
Keywords: Barrel medic; Medicago truncatula; Fabaceae; Strigolactone; Gigaspora margarita; Glomeromycota;

Effect of cadmium pollution on mobilization of embryo reserves in seedlings of six contrasted Medicago truncatula lines by Sondès Rahoui; Abdelilah Chaoui; Cécile Ben; Martina Rickauer; Laurent Gentzbittel; Ezzeddine El Ferjani (98-106).
Tolerance order and growth inhibition in Medicago truncatula genotypes could be correlated to reserves mobilization disorders, their leakage in germination media and MtMST expression alteration.Display OmittedSix Medicago truncatula genotypes differing in cadmium susceptibility were used to test the effect of this heavy metal on mineral, carbohydrate and amino acid supply in growing radicles. Cadmium treatment caused alteration of macronutrient (Ca and K), microelement (Fe, Zn and Cu), carbohydrate (total soluble sugars (TSS), glucose, fructose and sucrose) and free amino acid (FAAS) accumulations. These mobilization changes differed in the tested genotypes. Carbohydrates were determining to susceptible lines’ growth in control condition; free amino acids enabled tolerant lines to counteract cadmium intrusion. Transcriptional changes in response to cadmium treatment were analyzed on MtMST, a gene encoding a monosaccharide transport protein. A significant down-regulation was observed in the most susceptible line TN1.11. Glucose was over-consumed in tolerant lines. Thus, glucose metabolism integrity seems essential to maintain growth under cadmium exposure. Analysis of germination medium showed solute losses at the expense of suitable mobilization to the growing embryonic axis and highlights cadmium-triggered membrane alterations. FAAS and TSS leakages were reduced in tolerant lines while monosaccharide losses were accentuated in susceptible lines. This research work gave an overview of cadmium deleterious effects on biomass mobilization and membrane integrity. Carbon metabolism is shown to be primordial to enhance early embryonic growth and nitrogen metabolism is revealed to be crucial to establish seedling growth under cadmium stress.
Keywords: Amino acids; Cadmium; Carbohydrates; Medicago truncatula; Solute leakage; Sugar transport; Root growth;

n-Nonacosadienes from the marine haptophytes Emiliania huxleyi and Gephyrocapsa oceanica by Hideto Nakamura; Ken Sawada; Hiroya Araie; Iwane Suzuki; Yoshihiro Shiraiwa (107-113).
Cultured Emiliania huxleyi NIES837 and Gephyrocapsa oceanica NIES1315 abundantly produce C29 alkenes, including 2,20-nonacosadiene and 3,20-nonacosadiene identified.Display OmittedThe hydrocarbons in cultures of marine haptophytes Emiliania huxleyi NIES837 and Gephyrocapsa oceanica NIES1315 were analyzed, and nonacosadienes and hentriacontadienes were detected as the major compounds in both strains. C29 and C31 monoenes and di-, tri- and tetra-unsaturated C33 alkenes were also detected as minor compounds but not C37 and C38 alkenes. The positions of the double bonds in the C29 and C31 alkenes were determined by mass spectrometry of their dimethyl disulfide (DMDS) adducts. Among the four C29 alkenes identified, the most abundant isomer was 2,20-nonacosadiene, and the other three compounds were 1,20-nonacosadiene, 3,20-nonacosadiene and 9-nonacosene, respectively. Hitherto, 2,20-nonacosadiene and 3,20-nonacosadiene were unknown to be natural products. The double bond at the n-9 (ω9) position in these C29 alkenes is hypothesized to be derived from precursors of unsaturated fatty acids possessing an n-9 double bond, such as (9Z)-9-octadecenoic acid. Nonacosadienes have the potential for being used as distinct haptophyte biomarkers.
Keywords: Emiliania huxleyi; Gephyrocapsa oceanica; Haptophyte; Nonacosadiene; Noelaerhabdaceae; Long chain alkene; Biomarker;

Cell wall integrity, genotoxic injury and PCD dynamics in alfalfa saponin-treated white poplar cells highlight a complex link between molecule structure and activity by Stefania Paparella; Aldo Tava; Pinarosa Avato; Elisa Biazzi; Anca Macovei; Marco Biggiogera; Daniela Carbonera; Alma Balestrazzi (114-123).
Biological effects of Medicago sativa purified saponins were tested on proliferating white poplar cell suspension cultures. TEM analysis along with cytotoxicity and genotoxicity assays were performed.Display OmittedIn the present work, eleven saponins and three sapogenins purified from Medicago sativa were tested for their cytotoxicity against highly proliferating white poplar (Populus alba L.) cell suspension cultures. After preliminary screening, four saponins with different structural features in terms of aglycone moieties and sugar chains (saponin 3, a bidesmoside of hederagenin; saponins 4 and 5, monodesmoside and bidesmoside of medicagenic acid respectively, and saponin 10, a bidesmoside of zanhic acid) and different cytotoxicity were selected and used for further investigation on their structure–activity relationship. Transmission Electron Microscopy (TEM) analyses provided for the first time evidence of the effects exerted by saponins on plant cell wall integrity. Exposure to saponin 3 and saponin 10 resulted into disorganization of the outer wall layer and the effect was even more pronounced in white poplar cells treated with the two medicagenic acid derivatives, saponins 4 and 5. Oxidative burst and nitric oxide accumulation were common hallmarks of the response of white poplar cells to saponins. When DNA damage accumulation and DNA repair profiles were evaluated by Single Cell Gel Electrophoresis, induction of single and double strand breaks followed by effective repair was observed within 24 h. The reported data are discussed in view of the current issues dealing with saponin structure–activity relationship.
Keywords: Saponins; Cell wall; DNA damage response; Medicago sativa; Populus alba; Programmed cell death;

Isolation and SAR studies of bicyclic iminosugars from Castanospermum australe as glycosidase inhibitors by Atsushi Kato; Yuki Hirokami; Kyoko Kinami; Yutaro Tsuji; Shota Miyawaki; Isao Adachi; Jackie Hollinshead; Robert J. Nash; J.L. Kiappes; Nicole Zitzmann; Jin K. Cha; Russell J. Molyneux; George W.J. Fleet; Naoki Asano (124-131).
Fourteen iminosugars were isolated from Castanospermum australe. Our side-by-side comparison between bicyclic and corresponding monocyclic iminosugars revealed that inhibition potency and spectrum are clearly changed by their core structures against each enzyme.Display OmittedWe report the isolation and structural determination of fourteen iminosugars, containing five pyrrolizidines and five indolizidines, from Castanospermum australe. The structure of a new alkaloid was elucidated by spectroscopic methods as 6,8-diepi-castanospermine (13). Our side-by-side comparison between bicyclic and corresponding monocyclic iminosugars revealed that inhibition potency and spectrum against each enzyme are clearly changed by their core structures. Castanospermine (10) and 1-deoxynojirimycin (DNJ) have a common d-gluco configuration, and they showed the expected similar inhibition potency and spectrum. In sharp contrast, 6-epi-castanospermine (12) and 1-deoxymannojirimycin (manno-DNJ) both have the d-manno configuration but the α-mannosidase inhibition of 6-epi-castanospermine (12) was much better than that of manno-DNJ. 6,8-Diepi-castanospermine (13) could be regarded as a bicyclic derivative of talo-DNJ, but it showed a complete loss of α-galactosidase A inhibition. This behavior against α-galactosidase A is similar to that observed for 1-epi-australine (6) and altro-DMDP.
Keywords: Castanospermum australe; Castanospermine; Australine; Iminosugar; Glycosidase inhibitor; Structure–activity relationship;

Cytotoxic and natural killer cell stimulatory constituents of Phyllanthus songboiensis by Yulin Ren; Chunhua Yuan; Youcai Deng; Ragu Kanagasabai; Tran Ngoc Ninh; Vuong Tan Tu; Hee-Byung Chai; Djaja D. Soejarto; James R. Fuchs; Jack C. Yalowich; Jianhua Yu; A. Douglas Kinghorn (132-140).
Nine compounds, seven known, were isolated and identified from Phyllanthus songboiensis. An arylnaphthalene lignan was highly cytotoxic toward HT-29 cells, and a known phytosterol was found to stimulate natural killer cells in the presence of interleukin 12.Display OmittedA dichapetalin-type triterpenoid and a dibenzylbutyrolactone-type lignan, together with five known lignans, a known aromatic diterpenoid, and a known acylated phytosterol, were isolated from the aerial parts of Phyllanthus songboiensis, collected in Vietnam. Their structures were determined by interpretation of the spectroscopic data, and the inhibitory activity toward HT-29 human colon cancer cells of all isolates was evaluated by a cytotoxicity assay. The known arylnaphthalene lignan, (+)-acutissimalignan A, was highly cytotoxic toward HT-29 cells, with an IC50 value of 19 nM, but this compound was inactive as a DNA topoisomerase IIα (topo IIα) poison. The known phytosterol, (−)-β-sitosterol-3-O-β-d-(6-O-palmitoyl)glucopyranoside, was found to stimulate natural killer (NK) cells at a concentration of 10 μM in the presence of interleukin 12 (IL-12).
Keywords: Phyllanthus songboiensis; Phyllanthaceae; Lignan; Phytosterol; Cytotoxicity; DNA topoisomerase IIα; Natural killer cell;

Isoflavones with neuroprotective activities from fruits of Cudrania tricuspidata by Nguyen Tuan Hiep; Jaeyoung Kwon; Dong-Woo Kim; Bang Yeon Hwang; Hak-Ju Lee; Woongchon Mar; Dongho Lee (141-148).
Ten isoflavones, cudraisoflavones B–K, together with 27 known isoflavones, were isolated from the EtOAc soluble extract of the fruits of Cudrania tricuspidata. Six of these (compounds 79 and 1113) showed neuroprotective activity against 6-hydroxydopamine induced cell death in human neuroblastoma SH-SY5Y cells, with EC 50 values of 0.5–9.2 μM.Display OmittedTen isoflavones, cudraisoflavones B–K (110), together with 27 known isoflavones, were isolated from the EtOAc soluble extract of fruits of Cudrania tricuspidata. The structures of compounds 110 were elucidated on the basis of MS and NMR spectroscopic data, including 2D NMR experiments. Compounds 79 and three known (1113) compounds showed neuroprotective activity against 6-hydroxydopamine induced cell death in human neuroblastoma SH-SY5Y cells, with EC 50 values of 0.5–9.2 μM.
Keywords: Cudrania tricuspidata; Moraceae; Isoflavones; Neuroprotection; Parkinson’s disease; 6-OHDA; Human neuroblastoma SH-SY5Y;

13,14-Dihydrocoptisine – The genuine alkaloid from Chelidonium majus by Jana Paulsen; Mahdi Yahyazadeh; Sophie Hänsel; Maik Kleinwächter; Kerstin Ibrom; Dirk Selmar (149-153).
The genuine main alkaloid of greater celandine (Chelidonium majus) is 13,14-dihydrocoptisine and not coptisine as reported earlier. Structure was elucidated by one- and two-dimensional NMR techniques. In vivo, after tissue injuries, dihydrocoptisine is oxidized and converted to coptisine.Display OmittedThe genuine major benzylisoquinoline alkaloid occurring in the traditional medicinal plant greater celandine (Chelidonium majus L.) is 13,14-dihydrocoptisine and not – as described previously – coptisine. Structure of 13,14-dihydrocoptisine was elucidated. The discrepancy between the alkaloid pattern of the living plants and that of detached and dried leaves is due to the rapid and prompt conversion of 13,14-dihydrocoptisine to coptisine in the course of tissue injuries. Indeed, apart from the major alkaloid, some minor alkaloids might also be converted; this however is not in the centre of focus of this paper. This conversion is initiated by the change of pH. In vivo 13,14-dihydrocoptisine is localized in the acidic vacuoles, where it is stable. In contrast, in the neutral milieu, which results when vacuoles are destroyed in the course of tissue injuries, the genuine alkaloid is oxidized to yield coptisine. Accordingly, when alkaloids from C. majus should be analyzed, any postmortal conversion of 13,14-dihydrocoptisine has to be prevented.
Keywords: Coptisine; Dihydrocoptisine; Chelidonium majus; Greater celandine; Benzylisoquinoline alkaloids;

Dereplication-guided isolation of depsides thielavins S–T and lecanorins D–F from the endophytic fungus Setophoma sp. by Lívia S. de Medeiros; Lucas M. Abreu; Anita Nielsen; Hanne Ingmer; Thomas O. Larsen; Kristian F. Nielsen; Edson Rodrigues-Filho (154-162).
Depsides (17) were isolated from Setophoma sp., a fungus isolated from symptomless guava fruits. Their detection was assisted by HRMS based-dereplication. Their polyketide structures (e.g. 1, 4 and 5 b) were elucidated by means of HRMS/MS and NMR data analyses. Some compounds showed moderate activity against S. aureus.Display OmittedDereplication methodology using UHPLC–DAD–QTOFMS was applied during the metabolic profiling investigation of the endophyte Setophoma sp., a fungus isolated from symptomless guava fruits. The approach performed allowed a fast analysis of the microbial secondary metabolites. From this fungus, seven highly C-alkylated depsides were isolated and identified as polyketides thielavins S, T, U and V and lecanorins D, E and F. Their structures were elucidated through spectroscopic methods including NMR, HRMS and especially with assistance of HRMS/MS experiments. The compounds were tested for quorum sensing regulation activity in the virulence gene expression of Staphylococcus aureus, but no inhibitory effect was detected. Nevertheless, moderate antibacterial activity was encountered in three of tested depsides, particularly with thielavin T, whose MIC was 6.25 μg/mL against S. aureus.
Keywords: Setophoma sp.; Psidium guajava; Dereplication; Depsides; Antibacterial activity;

Sesquiterpene lactones from the aerial parts of Vernonia blumeoides growing in Nigeria by Abubakar Babando Aliyu; Brenda Moodley; Hafizah Chenia; Neil A. Koorbanally (163-168).
Four eudesmanolide sesquiterpene lactones were isolated from the aerial parts of Vernonia blumeoides. The absolute configuration of blumeoidolide-A was assigned by X-ray crystallography.Display OmittedFour eudesmanolide sesquiterpene lactones (14) were isolated from the aerial parts of Vernonia blumeoides used in Nigerian ethnomedicine for the treatment of diarrhea and malaria. Compound 1 demonstrated limited but interesting antibacterial activity against Bacillus, Staphylococcus and Streptococcus species. The crystal structure of 1 allowed the absolute configuration of the stereocentres in the molecule to be assigned.
Keywords: Eudesmanes; Sesquiterpene lactones; Antibacterial activity; Vernonia blumeoides;

Structures of eremophilane-type sesquiterpene glucosides, alticolosides A–G, from the Far Eastern endemic Ligularia alticola Worosch by Alexandra S. Silchenko; Anatoly I. Kalinovsky; Ludmila P. Ponomarenko; Sergey A. Avilov; Pelageya V. Andryjaschenko; Pavel S. Dmitrenok; Peter G. Gorovoy; Natalia Yu. Kim; Valentin A. Stonik (169-176).
Display OmittedSeven eremophilane-type sesquiterpene glucosides, alticolosides A–G, have been isolated from aerial parts of the endemic Far Eastern species Ligularia alticola Worosch. (Family Asteraceae) along with two known compounds, monoterpenoid glycoside (4S)-α-terpineol 8-O-β-d-glucopyranoside and norditerpenoid glycoside 7(8)-dihydro-β-ionone 3-O-β-d-glucopyranoside. Alticoloside D was identified with the earlier known 8-O-(β-d-glucopyranosyl)-2-oxo-eremophila-1(10),8,11-triene, but the stereostructure of the latter was revised on the basis of ROESY and CD data. All the glycosides are derivatives of new eremophilane-type aglycones, differing from known eremophilanes in details of planar and/or stereo structures except for the aglycone of alticoloside E.
Keywords: Ligularia alticola; Sesquiterpene eremophilane-type glycosides; Alticolosides; NMR;

On the formation of lignin polysaccharide networks in Norway spruce by Petri Oinonen; Liming Zhang; Martin Lawoko; Gunnar Henriksson (177-184).
Norway spruce hemicelluloses may form networks via lignin moieties through laccase cross linking in the early stages of lignificationDisplay OmittedIn this study we were mirroring suggested in vivo phenomena of lignin–hemicellulose complex formation in vitro, by cross-linking Norway spruce (Picea abies) galactoglucomannans, xylans and lignin moieties to high molecular weight complexes by laccase treatment. We were able to observe the oxidation and cross-linking of non-condensed guaiacyl-type phenolic moieties attached to both of the hemicelluloses by 31P NMR and size-exclusion chromatography. We suggest that hemicelluloses–lignin complexes form covalently linked structural units during the early stages of lignification via radical enzymatic cross-linking catalyzed by laccase. This work shows that the hemicellulose molecules in wood are covalently linked to two or more lignin units thereby making them suited for forming network structures.
Keywords: Norway spruce; Galactoglucomannan; Arabinoxylan; Lignin–carbohydrate networks; Laccase; Crosslinking; Lignification;