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Phytochemistry (v.69, #3)

Graphical contents list (pp. 577-584).

Biologically active compounds of semi-metals by Tomáš Řezanka; Karel Sigler (pp. 585-606).
The present state of knowledge in the chemistry of organic compounds of semi-metals from different organisms is reviewed. They include, e.g., the boron-containing antibiotics or the silicon compounds present in “silicate” bacteria. Arsenic is incorporated into arsenosugars, arsenobetaines or volatile methylated arsenic compounds, selenium is incorporated into selenocysteine that is found in some proteins. Other metalloids, i.e. the rare and toxic tellurium and the radioactive short-lived astatine, have no biological significance.Semi-metals (boron, silicon, arsenic and selenium) form organo-metal compounds, some of which are found in nature and affect the physiology of living organisms. They include, e.g., the boron-containing antibiotics aplasmomycin, borophycin, boromycin, and tartrolon or the silicon compounds present in “silicate” bacteria, relatives of the genus Bacillus, which release silicon from aluminosilicates through the secretion of organic acids. Arsenic is incorporated into arsenosugars and arsenobetaines by marine algae and invertebrates, and fungi and bacteria can produce volatile methylated arsenic compounds. Some prokaryotes can use arsenate as a terminal electron acceptor while others can utilize arsenite as an electron donor to generate energy. Selenium is incorporated into selenocysteine that is found in some proteins. Biomethylation of selenide produces methylselenide and dimethylselenide. Selenium analogues of amino acids, antitumor, antibacterial, antifungal, antiviral, anti-infective drugs are often used as analogues of important pharmacological sulfur compounds. Other metalloids, i.e. the rare and toxic tellurium and the radioactive short-lived astatine, have no biological significance.

Keywords: Semi-metals; Boron; Silicon; Arsenic; Selenium; Organometallic compounds; Bacteria; Fungi; Higher plants

Sesquiterpene lactones as chemotaxonomic markers in genus Anthemis by Jordanka D. Staneva; Milka N. Todorova; Ljuba N. Evstatieva (pp. 607-618).
A chemotaxonomic discussion on the basis of the lactone profile of 18 species from genus Anthemis is presented. A. macedonica is suggested to be transferred from sect. Anthemis to sect. Hiorthia.Sesquiterpene lactones isolated from the genus Anthemis are used as chemotaxonomic markers. The obtained results support with some exceptions the botanical classification in Flora Europaea. Discrepancy between the lactone profile, cluster analysis and classification of A. melampodina, A. macedonica and A. austriaca in the genus Anthemis is discussed. The lactone composition of the undescribed as an European species A. plutonia correlates well with the guaianolide containing group of sect. Hiorthia.

Keywords: Genus; Anthemis; Asteraceae; Chemotaxonomy; Sesquiterpene lactones

Purification and characterization of malonyl-coenzyme A: 21-hydroxypregnane 21- O-malonyltransferase ( Dp21MaT) from leaves of Digitalis purpurea L. by Serge Philibert Kuate; Rodrigo M. Pádua; Wilhelm F. Eisenbeiss; Wolfgang Kreis (pp. 619-626).
Malonyl-coenzyme A: 21-Hydroxypregnane 21- O-malonyltransferase catalyzing the transfer of a malonyl moiety to the 21-hydroxy group of 21-hydroxypregnanes has been purified and characterized. This enzyme probably initiates butenolide ring formation in cardenolide biosynthesis.With respect to the cardenolide pathway and the characterization of enzymes involved in the formation of cardenolides, a malonyltransferase, termed malonyl-coenzyme A: 21-hydroxypregnane 21- O-malonyltransferase ( Dp21MaT) has been purified. The enzyme catalyses the transfer of the malonyl moiety from malonyl-coenzyme A to 21-hydroxypregnane substrates. Malonyltransferase activity was checked in several potential starting materials including fresh leaves and cell suspension cultures from different plants. Fresh Digitalis purpurea L. leaves turned out to be the best enzyme source. The purification protocol included ammonium sulphate precipitation, hydrophobic interaction chromatography on Phenylsepharose 6 FF, ion exchange chromatography on Source 30 Q, affinity chromatography on Cibacron Blue 3GA and gel filtration on Superdex 75. Gel filtration and native SDS–PAGE analysis showed that Dp21MaT exists as a monomer with a molecular mass of 27kDa. Its p I, as determined by isoelectric focusing, was 4.66. The enzyme showed maximal activity at pH 6.5 when incubated at 42°C. The energy of activation was 29.28kJmol−1, whereas that of inactivation was 48.57kJmol−1. Dp21MaT was purified 252-fold with a yield of about 1%. Hanes plots of kinetic data indicated Km values of 99μM ( Vmax 47.57μkatkg−1) and 28.44μM ( Vmax 39.4μkatkg−1 protein) for 3β-benzoyloxy-5β-pregnane-14β,21-dihydroxy-20-one and malonyl-CoA, respectively.

Keywords: Digitalis purpurea; Plantaginaceae; Enzyme purification; Malonyl-coenzyme A; 21-Hydroxypregnane 21-; O; -malonyltransferase; 21-; O; -Hydroxypregnanes; Cardenolides; Cardiac glycosides

Spring cabbage peroxidases – Potential tool in biocatalysis and bioelectrocatalysis by Anna Belcarz; Grazyna Ginalska; Barbara Kowalewska; Pawel Kulesza (pp. 627-636).
Two peroxidase fractions were separated and partially purified from spring cabbage heads. Their characterization and potential practical application in bioelectrocatalytic systems for H2O2 reduction were described.Two fractions of peroxidase activity, cationic Px-cat and anionic Px-ani, were isolated and partially purified (143.5- and 5.49-fold, respectively) from homogenate of spring cabbage heads. Optimum pH for both fractions is 6.0; however, Px-cat is almost equally active at neutral pH (7.0) while Px-ani reveals high activity in more acidic pHs (with 60% of maximum activity at pH 3.0). Optimal temperature for both fractions was 40°C. Px-ani possessed much higher thermal stability at 40–50°C (60% of remaining activity after 144h of incubation) than Px-cat. The peroxidases remained fully active during 4 weeks of storage at 4°C. Kinetic studies revealed that Px-cat and Px-ani had lower apparent Km values for ABTS (0.0377 and 0.0625mM) and o-dianisidine (0.357 and 0.286mM) than for guaiacol (6.41 and 13.89mM). The best substrate for Px-cat was pyrogallol and for Px-ani– o-dianisidine. Px-cat immobilized on polyanionic PyBA-modified carbon electrode was found to produce linear repetitive signals upon consecutive additions of hydrogen peroxide during at least 1-week period and to work effectively under buffered and non-buffered conditions. These properties were comparable with those of commercially available horseradish peroxidase. Stability of the hybrid bioelectrocatalytic film and low costs of extraction and partial purification of Px-cat make it a highly promising enzyme for practical applications, including construction of bioelectrodes.

Keywords: Spring cabbage peroxidase; Enzyme stability; Electrodes; Carbon nanotubes; Hydrogen peroxide reduction

Quantitative analysis of auxin-regulated proteins from basal part of leaf sheaths in rice by two-dimensional difference gel electrophoresis by Fang Shi; Hironori Takasaki; Setsuko Komatsu (pp. 637-646).
To identify the effects of auxin on rice root formation, proteins induced by exogenous addition of auxin to rice seedlings were analyzed by a proteomic approach. Root formation by rice seedlings was promoted by 2,4-D and repressed by PCIB. Based on proteomic analyses, mitochondrial complex I subunit is part of the downstream signal cascade of PCIB, whereas myosin heavy chain, mitochondrial [Mn]SOD and EF-1β′ are involved in the 2,4-D signal cascade but are probably upstream of PCIB.To identify the effects of auxin on rice root formation, proteins induced by exogenous addition of auxin to rice seedlings were analyzed by a proteomic approach. Root formation by rice seedlings was promoted by 0.45μM 2,4-dichlorophenoxyacetic acid (2,4-D) and repressed by 60μM p-chlorophenoxyisobutyric acid (PCIB). Proteins extracted from the basal part of leaf sheaths of rice seedlings treated with 2,4-D or PCIB for 48h were labeled with Cy3 and Cy5, and separated by two-dimensional polyacrylamide gel electrophoresis. Out of nine proteins up-regulated by 2,4-D and down-regulated by PCIB, five proteins showing significant difference in abundance were used for expression analysis at the transcript abundance level. Transcript abundance of the mitochondrial complex I subunit slightly increased with 2,4-D treatment and were repressed by PCIB. The transcript abundance of EF-1β′, myosin heavy chain and mitochondrial [Mn]SOD increased with 2,4-D treatment but did not decrease with PCIB. The transcript abundance of aldehyde dehydrogenase was not effected by 2,4-D or PCIB. These results indicate that mitochondrial complex I subunit is part of the downstream signal cascade of PCIB, whereas myosin heavy chain, mitochondrial [Mn]SOD and EF-1β′ are involved in the 2,4-D signal cascade but are probably upstream of PCIB.

Keywords: Rice; Auxin; 2,4-D; PCIB; Oryza sativa; Gramineae; Proteomics; RT-PCRAbbreviations; 2,4-D; 2,4-dichlorophenoxyacetic acid; PCIB; p; -chlorophenoxyisobutyric acid; 2D-PAGE; two-dimensional polyacrylamide gel electrophoresis; 2D-DIGE; two-dimensional difference gel electrophoresis; SOD; superoxide dismutase; RuBisCO; ribulose-1,5-bisphosphate carboxylase/oxygenase; EF-1β′; elongation factor-1β′

An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.) by K.B. Devaraj; Lalitha R. Gowda; V. Prakash (pp. 647-655).
An aspartic protease has been purified from the latex of Ficus racemosa and characterized. The molecular mass, single isoform, pH optima and stability of the protease are unique and differ from other known ficins.The most extensively studied ficins have been isolated from the latex of Ficus glabrata and Ficus carica. However the proteases (ficins) from other species are less known. The purification and characterization of a protease from the latex of Ficus racemosa is reported. The enzyme purified to homogeneity is a single polypeptide chain of molecular weight of 44,500±500Da as determined by MALDI-TOF. The enzyme exhibited a broad spectrum of pH optima between pH 4.5–6.5 and showed maximum activity at 60±0.5°C. The enzyme activity was completely inhibited by pepstatin-A indicating that the purified enzyme is an aspartic protease. Far-UV circular dichroic spectra revealed that the purified enzyme contains predominantly β-structures. The purified protease is thermostable. The apparent Tm, (mid point of thermal inactivation) was found to be 70±0.5°C. Thermal inactivation was found to follow first order kinetics at pH 5.5. Activation energy ( Ea) was found to be 44.0±0.3kcalmol−1. The activation enthalpy (Δ H), free energy change (Δ G) and entropy (Δ S) were estimated to be 43±4kcalmol−1, −26±3kcalmol−1 and 204±10calmol−1K−1, respectively. Its enzymatic specificity studied using oxidized B chain of insulin indicates that the protease preferably hydrolyzed peptide bonds C-terminal to glutamate, leucine and phenylalanine (at P1 position). The broad specificity, pH optima and elevated thermal stability indicate the protease is distinct from other known ficins and would find applications in many sectors for its unique properties.

Keywords: Ficus racemosa; Moraceae; Aspartic protease; Plant proteases; Endopeptidases; Ficin

Hevea latex lectin binding protein in C-serum as an anti-latex coagulating factor and its role in a proposed new model for latex coagulation by Rapepun Wititsuwannakul; Piyaporn Pasitkul; Pattavuth Jewtragoon; Dhirayos Wititsuwannakul (pp. 656-662).
A protein specific for Hevea latex lectin binding was isolated and purified from C-serum of fresh latex. Its biochemical properties and physiological involvement in a new model for latex coagulation is presented.A distinct protein specifically recognized by its strong interaction with Hevea latex lectin (HLL) was detected in the aqueous C-serum fraction of centrifuged fresh latex. This C-serum lectin binding protein (CS-HLLBP) exhibited strong inhibition of HLL-induced hemagglutination. The CS-HLLBP was purified to homogeneity by a protocol that included ammonium sulfate fractionation, size exclusion and ion exchange chromatography. The purified CS-HLLBP had a specific HI titer of 0.23μgml−1. Its Mrs analyzed by SDS–PAGE was ca. 40kDa and that by gel filtration was ca. 204kDa. It has a p I value of 4.7, an optimum activity between pH 6 and10 and was heat stable up to 50°C. The HI activity of CS-HLLBP was abolished upon treatment with chitinase. The CS-HLLBP inhibited HLL-induced rubber particle aggregation in a dose dependent manner. A highly positive correlation between CS-HLLBP activity and rubber yield per tapping was found. The correlations for fresh latex ( r=0.98, P<0.01) and dry rubber ( r=0.95, P<0.01) were both highly significant. This indicated that the CS-HLLBP might be used as a reliable marker for the mass screening of young seedlings to identify and select clones with potential to be superior producers of rubber. A latex anti-coagulating role of the CS-HLLBP is proposed. The findings described in this 3 paper series have been used to propose a new model of rubber latex coagulation that logically describes roles for the newly characterized latex lectin and the two lectin binding proteins.

Keywords: Hevea brasiliensis; Euphorbiaceae; Rubber latex; C-serum; Lectin; Lectin binding protein; α-Globulin; Latex flow; Anti-coagulating factor; Latex coagulation

ESP and ESM1 mediate indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate in Arabidopsis by Meike Burow; Zhi-Yong Zhang; James A. Ober; Virginia M. Lambrix; Ute Wittstock; Jonathan Gershenzon; Daniel J. Kliebenstein / (pp. 663-671).
Multiple proteins function to control the production of indol-3-acetonitrile during indole glucosinolate activation/hydrolysis.Glucosinolates are plant secondary metabolites that act as direct defenses against insect herbivores and various pathogens. Recent analysis has shown that methionine-derived glucosinolates are hydrolyzed/activated into either nitriles or isothiocyanates depending upon the plants genotype at multiple loci. While it has been hypothesized that tryptophan-derived glucosinolates can be a source of indole-acetonitriles, it has not been explicitly shown if the same proteins control nitrile production from tryptophan-derived glucosinolates as from methionine-derived glucosinolates. In this report, we formally test if the proteins involved in controlling aliphatic glucosinolate hydrolysis during tissue disruption can control production of nitriles during indolic glucosinolate hydrolysis. We show that myrosinase is not sufficient for indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate and requires the presence of functional epithospecifier protein in planta and in vitro to produce significant levels of indol-3-acetonitrile. This reaction is also controlled by the Epithiospecifier modifier 1 gene. Thus, like formation of nitriles from aliphatic glucosinolates, indol-3-acetonitrile production following tissue disruption is controlled by multiple loci raising the potential for complex regulation and fine tuning of indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate.

Keywords: Arabidopsis thaliana; Brassicaceae; Quantitative genetics; Biochemical assay; Indole glucosinolate; Indol-3-acetonitrile; Indol-3-carbinol; Epithiospecifier protein; Epithiospecifier modifier 1 protein

Influence of electron transport proteins on the reactions catalyzed by Fusarium fujikuroi gibberellin monooxygenases by Claudia Troncoso; José Cárcamo; Peter Hedden; Bettina Tudzynski; M. Cecilia Rojas (pp. 672-683).
Products formed by GA14 synthase and gibberellin 20-oxidase in Fusarium fujikuroi mutants that lack cytochrome P450 reductase (CPR) are qualitatively and quantitatively different from those produced in CPR-containing strains. Interaction of these P450s with cyt b5:cyt b5 reductase in the mutants would account for the differences found.The multifunctional cytochrome P450 monooxygenases P450-1 and P450-2 from Fusarium fujikuroi catalyze the formation of GA14 and GA4, respectively, in the gibberellin (GA)-biosynthetic pathway. However, the activity of these enzymes is qualitatively and quantitatively different in mutants lacking the NADPH:cytochrome P450 oxidoreductase (CPR) compared to CPR-containing strains. 3β-Hydroxylation, a major P450-1 activity in wild-type strains, was strongly decreased in the mutants relative to oxidation at C-6 and C-7, while synthesis of C19-GAs as a result of oxidative cleavage of C-20 by P450-2 was almost absent whereas the C-20 alcohol, aldehyde and carboxylic acid derivatives accumulated. Interaction of the monooxygenases with alternative electron transport proteins could account for these different product distributions. In the absence of CPR, P450-1 activities were NADH-dependent, and stimulated by cytochrome b5 or by added FAD. These properties as well as the decreased efficiency of P450-1 and P450-2 in the mutants are consistent with the participation of cytochrome b5:NADH cytochrome b5 reductase as redox partner of the gibberellin monooxygenases in the absence of CPR. We provide evidence, from either incubations of GA12 (C-20 methyl) with cultures of the mutant suspended in [18O]H2O or maintained under an atmosphere of [18O]O2:N2 (20:80), that GA15 (C-20 alcohol) and GA24 (C-20 aldehyde) are formed directly from dioxygen and not from hydrolysis of covalently enzyme-bound intermediates. Thus these partially oxidized GAs correspond to intermediates of the sequential oxidation of C-20 catalyzed by P450-2.

Keywords: Fusarium fujikuroi; Gibberellin biosynthesis; GA; 14; synthase; Gibberellin 20-oxidase; P450 monooxygenases; Electron transport proteins

Lipid characterization of a wrinkled sunflower mutant by Mónica Venegas-Calerón; Enrique Martínez-Force; Rafael Garcés (pp. 684-691).
This mutant seed has a wrinkled phenotype, with reduced content of storage triacylglycerols, normal phospholipids contents and increase content of palmitic and deficient biosynthesis of linolenic acid during seed germination.As part of a sunflower mutagenesis program carried out to obtain lines with fatty acid profiles in their oils, the half-palmitic CAS-7 line, with ca. 14% palmitic acid content, was isolated. Attempts to obtain a homozygotic line proved to be futile due to the lack of growth of the seedlings 10–12 days after germination. At this age, the seedlings stop growing, displayed a lack of chlorophyll and poor linolenic acid content, a fatty acid intimately linked to photosynthetic membranes. Accordingly, this line has only been maintained through heterozygotic seeds. Likewise, the cotyledons of seeds from this line with medium levels of palmitic acid present a characteristic wrinkled phenotype. In the oil of these seeds, the triacylglycerol content displayed a reduction of approximately 57% with respect to the control line, although a similar reduction was not observed in the polar lipids. Furthermore this mutant has 40.0% of trilinolein, the higher content found until today in sunflower seeds. These data indicate that the CAS-7 mutant possesses a multiple phenotype having a reduced triacylglycerol seed content, a modified intraplastidial fatty acid synthesis, together with a seedling blocked growth and poor green colour and reduced chloroplast development.

Keywords: Helianthus annuus; Fatty acids; Lipid; Mutant; Seed; Wrinkled; Linolenic; PalmiticAbbreviations; ACP; acyl carrier protein; DAS; days after sowing; FAS; fatty acid synthetase; KAS; ß-Keto-acyl-ACP synthase; SAD; stearoyl-ACP desaturase; TAG; triacylglycerol

Cell wall invertase-deficient miniature1 kernels have altered phytohormone levels by Sherry LeClere; Eric A. Schmelz; Prem S. Chourey (pp. 692-699).
Kernels of the Zea mays cell wall invertase (INCW)-deficient miniature1 seed mutant display dramatic decreases in indole-3-acetic acid (IAA) levels and alterations in salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA) levels, indicating that alterations in INCW-mediated sucrose cleavage affect downstream hormone homeostasis.The Zea mays (maize) miniature1 ( Mn1) locus encodes the cell wall invertase INCW2, which is localized predominantly in the basal endosperm transfer layer (BETL) of developing kernels and catalyzes conversion of sucrose into glucose and fructose. Mutations in Mn1 result in numerous changes that include a small kernel phenotype resulting from both decreased cell size and number. To explore the pleiotropic effects of this mutation, we investigated the levels of indole-3-acetic acid (IAA), abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA) in basal regions, upper regions, and embryos of developing kernels in the inbred line W22. We measured phytohormones from 6 to 28 days after pollination (DAP) in wild type (WT) and two alleles of mn1, mn1–1 and mn1–89. IAA was the predominant hormone in kernels, with WT levels of free IAA accumulating over time to more than 2μg/g of fresh weight. Kernels of mn1–1 accumulated up to 10-fold less IAA than WT, and levels of IAA sugar conjugates were similarly reduced. Although less abundant, differences were also observed in levels of ABA, JA, and SA between WT and the mn1 alleles. SA levels were increased by as much as 10-fold in mn1–1, and mn1–89 displayed intermediate SA levels at most timepoints. These findings indicate that invertase-mediated sucrose cleavage directly or indirectly regulates the levels of key plant hormones during seed development.

Keywords: Zea mays; Gramineae; Maize; Phytohormone analysis; Indole-3-acetic acid; Salicylic acid; Jasmonic acid; Abscisic acid; Cell wall invertase; Miniature1; INCW2

Accumulation of trans-piceid in sorghum seedlings infected with Colletotrichum sublineolum by Christine K.Y. Yu; Chun-Hat Shih; Ivan K. Chu; Clive Lo (pp. 700-706).
Using LC-MS in precursor ion scan mode, trans-piceid was identified as the major stilbene metabolite in sorghum seedlings infected with the anthracnose pathogen Colletotrichum sublineolum. However, trans-piceid alone may not be a significant defense component against this pathogen. This is the first report of stilbene accumulation in sorghum.Sorghum SbSTS1, a pathogen inducible gene, was previously demonstrated to encode an enzyme with stilbene synthase activity. In this study, we attempt to identify the stilbene derivatives that accumulate in infected sorghum seedlings after inoculation with the anthracnose pathogen Colletotrichum sublineolum. Scanning for precursor ions that produced the common stilbene aglycones as diagnostic ions was performed in a triple quadrupole mass spectrometer. It was found that infected sorghum seedlings accumulated trans-piceid as the major stilbene metabolite together with an unknown resveratrol derivative. Time-course accumulation of trans-piceid was examined in two sorghum cultivars, DK18 and DK77, which are resistant and susceptible to C. sublineolum, respectively. In both cultivars, trans-piceid was not detected until 48h after inoculation, consistent with the late induction of SbSTS1 reported previously in infected sorghum plants. The levels of trans-piceid detected in DK77 seedlings were approximately three times the levels detected in DK18 seedlings at 120h after inoculation. In vitro assays demonstrated that trans-piceid did not exhibit significant toxicity on conidial germination and mycelial growth of C. sublineolum. Hence trans-piceid alone may not represent an important defense component against the anthracnose pathogen in sorghum seedlings.

Keywords: Anthracnose; Colletotrichum sublineolum; Sorghum bicolor; Poaceae; Precursor ion scan; Stilbene glycosides; Stilbene synthase; trans; -Piceid

Detoxification of 2,4-dichlorophenol by the marine microalga Tetraselmis marina by Dimitris Petroutsos; Petros Katapodis; Martina Samiotaki; George Panayotou; Dimitris Kekos (pp. 707-714).
Xenobiotic chlorinated phenols have been found in fresh and marine waters and are toxic to many aquatic organisms. The marine microalga Tetraselmis marina has been found to tolerate and metabolize 2,4-dichlorophenol. The detoxification pathway has been elucidated.Xenobiotic chlorinated phenols have been found in fresh and marine waters and are toxic to many aquatic organisms. Metabolism of 2,4-dichlorophenol (2,4-DCP) in the marine microalga Tetraselmis marina was studied. The microalga removed more than 1mM of 2,4-DCP in a 2l photobioreactor over a 6day period. Two metabolites, more polar than 2,4-DCP, were detected in the growth medium by reverse phase HPLC and their concentrations increased at the expense of 2,4-DCP. The metabolites were isolated by a C8 HPLC column and identified as 2,4-dichlorophenyl-β-d-glucopyranoside (DCPG) and 2,4-dichlorophenyl-β-d-(6- O-malonyl)-glucopyranoside (DCPGM) by electrospray ionization-mass spectrometric analysis in a negative ion mode. The molecular structures of 2,4-DCPG and 2,4-CPGM were further confirmed by enzymatic and alkaline hydrolyses. Thus, it was concluded that the major pathway of 2,4-DCP metabolism in T. marina involves an initial conjugation of 2,4-DCP to glucose to form 2,4-dichlorophenyl-β-d-glucopyranoside, followed by acylation of the glucoconjugate to form 2,4-dichlorophenyl-β-d-(6- O-malonyl)-glucopyranoside. The microalga ability to detoxify dichlorophenol congeners other than 2,4-DCP was also investigated. This work provides the first evidence that microalgae can use a combined glucosyl and malonyl transfer to detoxify xenobiotics such as dichlorophenols.

Keywords: Tetraselmis marina; Marine microalgae; Xenobiotic metabolism; Detoxification; Chlorophenols

Proteomic analysis of rice defense response induced by probenazole by Yu-Zu Lin; Huai-Yi Chen; Ruby Kao; Shih-Pai Chang; Su-Jein Chang; Erh-Min Lai (pp. 715-728).
We report the first proteome of a rice defense response induced by a plant activator, PBZ. Eleven unique proteins from 9 PBZ-regulated spots were identified. We proposed that the identified PBZ-induced proteins PAL, COMT, and GSTU17 may confer PBZ-induced disease resistance via such functions as biosynthesis and transport of flavonoid-type phytoalexin and/or lignin biogenesis.Here, we report the first proteomic analysis of rice defense response induced by probenazole (PBZ), an agricultural chemical that has been widely used to protect rice plants from rice blast and the bacterial blight pathogen. Two-dimensional gel electrophoresis (2-DE) was utilized to identify a total of 40 protein spots including 9 protein spots that are up-regulated by PBZ and 31 abundant protein spots. A total of 11 unique proteins from these 9 spots were identified by LC–MS/MS, and the majority of them were classified and/or possessed orthologs in defense-related functions. Five protein spots with only one protein species identified in each spot appear to be PBZ-regulated proteins. They are a putative glutathione S-transferase GSTU17, a putative phenylalanine ammonia-lyase (PAL, XP_466843), a putative caffeic acid 3- O-methyltransferase (COMT), a putative NADH-ubiquinone oxidoreductase, and a putative glucose-1-phosphate adenyltransferase. However, the other six protein species identified from the remaining four protein spots could not be conclusively described as PBZ-regulated proteins due to either the co-migration of two protein species in one spot or the presence of one protein species in two spots. Through real-time reverse transcription polymerase chain reaction (RT-PCR), it was determined that PAL (XP_466843) is likely regulated at the protein level, whereas GSTU17 and COMT were regulated at the mRNA level after PBZ application. Interestingly, the mRNA transcripts of two PAL paralogs were found to be up-regulated by PBZ. We propose that PAL, COMT, and GSTU17 are likely to confer PBZ-induced disease resistance via such functions as biosynthesis and transport of flavonoid-type phytoalexin and/or lignin biogenesis.

Keywords: Rice; Oryza sativa; Graminaceae; Defense; Disease resistance; Probenazole; Proteomics

Steroidal glycosides from the rhizomes of Ruscus hypophyllum by Yoshihiro Mimaki; Tsukasa Aoki; Maki Jitsuno; Ceyda Sibel Kiliç; Maksut Coşkun (pp. 729-737).
Seven steroidal glycosides, along with one known glycoside, were isolated from the rhizomes of Ruscus hypophyllum (Liliaceae).Seven steroidal glycosides, along with one known glycoside, were isolated from the rhizomes of Ruscus hypophyllum (Liliaceae). Comprehensive spectroscopic analysis, including 2D NMR spectroscopy, and the results of acid hydrolysis allowed the chemical structures of the compounds to be assigned as (23 S,25 R)-23-hydroxyspirost-5-en-3β-yl O-α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranoside (1), 1β-hydroxyspirosta-5,25(27)-dien-3β-yl O-α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranoside (2), (22 S)-16β,22-dihydroxycholest-5-en-3β-yl O-α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranoside (3), (22 S)-16β-[(β-d-glucopyranosyl)oxy]-22-hydroxycholest-5-en-3β-yl O-α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranoside (4), (22 S)-16β-[(β-d-glucopyranosyl)oxy]-22-hydroxycholest-5-en-3β-yl β-d-glucopyranoside (5), (22 S)-16β-[(β-d-glucopyranosyl)oxy]-3β,22-dihydroxycholest-5-en-1β-yl O-α-l-rhamnopyranosyl-(1→2)-(3,4-di- O-acetyl-β-d-xylopyranoside) (6), and (22 S)-16β-[(β-d-glucopyranosyl)oxy]-3β,22-dihydroxycholest-5-en-1β-yl O-α-l-rhamnopyranosyl-(1→2)- O-[β-d-xylopyranosyl-(1→3)]-β-d-xylopyranoside (7), respectively. This is the first isolation of a series of cholestane glycosides from a Ruscus species.

Keywords: Ruscus hypophyllum; Liliaceae; Glycosides; Spirostanol saponins; Cholestane glycosides

Antifungal and antioxidant activities of the phytomedicine pipsissewa, Chimaphila umbellata by Imelda J. Galván; Nadereh Mir-Rashed; Matthew Jessulat; Monica Atanya; Ashkan Golshani; Tony Durst; Philippe Petit; Virginie Treyvaud Amiguet; Teun Boekhout; Richard Summerbell; Isabel Cruz; John T. Arnason; Myron L. Smith (pp. 738-746).
Demonstration of antifungal and antioxidant effects supports the traditional use of pipsissewa in treating skin ailments. Chemical-genetic profiling with a library of yeast deletion mutants indicated that the principal antifungal constituent, chimaphilin, had a complex mode of action, affecting a protein–protein interaction network including 26% of the genes in the profile.Bioassay-guided fractionation of Chimaphila umbellata (L.) W. Bart (Pyrolaceae) ethanol extracts led to the identification of 2,7-dimethyl-1,4-naphthoquinone (chimaphilin) as the principal antifungal component. The structure of chimaphilin was confirmed by1H and13C NMR spectroscopy. The antifungal activity of chimaphilin was evaluated using the microdilution method with Saccharomyces cerevisiae (0.05mg/mL) and the dandruff-associated fungi Malassezia globosa (0.39mg/mL) and Malassezia restricta (0.55mg/mL). Pronounced antioxidant activity of C. umbellata crude extract was also identified using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, suggesting this phytomedicine has an antioxidant function in wound healing. A chemical-genetic profile was completed with chimaphilin using ∼4700 S. cerevisiae gene deletion mutants. Cellular roles of deleted genes in the most susceptible mutants and secondary assays indicate that the targets for chimaphilin include pathways involved in cell wall biogenesis and transcription.

Keywords: Chimaphila umbellata; Malassezia; Antifungal mode of action; Antioxidant activity; Chemical-genetic profile; 2,7-Dimethyl-1,4-naphthoquinone

Bioactive monoterpene glycosides conjugated with gallic acid from the leaves of Eucalyptus globulus by Tatsuya Hasegawa; Fumihide Takano; Takanobu Takata; Masato Niiyama; Tomihisa Ohta (pp. 747-753).
Two monoterpene glycosides conjugated with gallic acid [globulusin A (1) and B (2)], together with four known compounds, were isolated from hot-water extracts of the leaves of Eucalyptus globulus. Globulusin A (1) and eucaglobulin (4) exhibited significant antioxidant, anti-inflammatory and anti-melanogenesis activity.Two monoterpene glycosides, conjugated with gallic acid [globulusin A (1) and B (2)], together with four known compounds, cypellocarpin A (3), eucaglobulin (4), cuniloside (5) and (1 S, 2 S, 4 R)- trans-2-hydroxy-1,8-cineole β-d-glucopyranoside (6), were isolated from hot-water extracts of the leaves of Eucalyptus globulus. The structures of compounds1 and2 were determined by 1D, 2D NMR and MS spectroscopic analyses. The absolute stereochemistry of1 was determined by correlating the spectroscopic data with those of synthetic compound6 with a known configuration. Globulusin A (1) and B (2), cypellocarpin A (3) and eucaglobulin (4), scavenged DPPH free radicals and globulusin A (1) showed a higher antioxidant activity than the other tested compounds, with an IC50 of 3.8μM. Globulusin A (1) and eucaglobulin (4) concentration-dependently suppressed inflammatory cytokine production, tumor-necrosis factor-α and interleukin-1β in cultured human myeloma THP-1 cells co-stimulated with phorbol myristate acetate. These compounds also inhibited melanogenesis in cultured murine melanoma B16F1 cells, without any significant cytotoxicity. These results suggested that globulusin A (1) and eucaglobulin (4), which were isolated as antioxidants from E. globulus, also had anti-inflammatory as well as anti-melanogenesis activity.

Keywords: Eucalyptus globulus; Myrtaceae; Monoterpene; Glucosides; Gallic acid

Xanthones with quinone reductase-inducing activity from the fruits of Garcinia mangostana (Mangosteen) by Young-Won Chin; Hyun-Ah Jung; Heebyung Chai; William J. Keller; A. Douglas Kinghorn (pp. 754-758).
Four quinone reductase-inducing xanthones, including two xanthones (1 and2), were isolated from Garcinia mangostana.Bioactivity-guided fractionation of a dichloromethane-soluble extract of Garcinia mangostana fruits has led to the isolation and identification of five compounds, including two xanthones, 1,2-dihydro-1,8,10-trihydroxy-2-(2-hydroxypropan-2-yl)-9-(3-methylbut-2-enyl)furo[3,2- a]xanthen-11-one (1) and 6-deoxy-7-demethylmangostanin (2), along with three known compounds, 1,3,7-trihydroxy-2,8-di-(3-methylbut-2-enyl)xanthone (3), mangostanin (4), and α-mangostin (5). The structures of compounds1 and2 were determined from analysis of their spectroscopic data. All isolated compounds in the present study together with eleven other compounds previously isolated from the pericarp of mangosteen, were tested in an in vitro quinone reductase-induction assay using murine hepatoma cells (Hepa 1c1c7) and an in vitro hydroxyl radical antioxidant assay. Of these, compounds14 induced quinone reductase (concentration to double enzyme induction, 0.68–2.2μg/mL) in Hepa 1c1c7 cells and γ-mangostin (6) exhibited hydroxyl radical-scavenging activity (IC50, 0.20μg/mL).

Keywords: Garcinia mangostana; Clusiaceae; 1,2-Dihydro-1,8,10-trihydroxy-2-(2-hydroxypropan-2-yl)-9-(3-methylbut-2-enyl)furo[3,2-; a; ]xanthen-11-one; 2,3-Dihydro-4,7-dihydroxy-2-(2-hydroxypropan-2-yl)-6-(3-methylbut-2-enyl)furo[3,2-; b; ]xanthen-5-one; (6-deoxy-7-demethylmangostanin); Quinone reductase induction; Hydroxyl radical-scavenging activity

Nematicidal prenylated flavanones from Phyllanthus niruri by N.A. Shakil; Pankaj; J. Kumar; R.K. Pandey; D.B. Saxena (pp. 759-764).
The hexane extract of Phyllanthus niruri plant, on rigorous column chromatography, yielded two prenylated flavanones, viz. 8-(3-methyl-but-2-enyl)-2-phenyl chroman-4-one (1) and 2-(4-hydroxyphenyl)-8-(3-methyl-but-2-enyl)-chroman-4-one (2). These were evaluated for nematicidal activity against two nematodes, i.e. root-knot ( Meloidogyne incognita) and reniform ( Rotylenchulus reniformis). The LC50 values ranged between 3.3 to 14.5ppm as compared to 3.1ppm for standard carbofuran.Two prenylated flavanones have been isolated from the hexane extract of Phyllanthus niruri plant. The structure of these flavanones were established as 8-(3-Methyl-but-2-enyl)-2-phenyl chroman-4-one (1) and 2-(4-hydroxyphenyl)-8-(3-methyl-but-2-enyl)-chroman-4-one (2) on the basis of spectral analysis. These were evaluated for nematicidal activity against root-knot, Meloidogyne incognita, and reniform, Rotylenchulus reniformis, nematodes. Compound2 exhibited nematicidal activity at par with the standard carbofuran (LC50 3.3 and 3.1ppm, respectively) when tested against reniform nematode. The LC50 value against root-knot nematode was found to be 14.5ppm. Compound1 however, showed moderate activity against both the test nematodes.

Keywords: Phyllanthus niruri; Flavanones; Meloidogyne incognita; Rotylenchulus reniformis; Nematodes; Euphorbiaceae

Biotransformation of myrislignan by rat liver microsomes in vitro by Fei Li; Xiu-Wei Yang (pp. 765-771).
Myrislignan (1) is a major acyclic neolignan in seeds of Myristica fragrans. We investigated the biotransformation of myrislignan by rat liver microsomes in vitro. Seven metabolites were produced by rat liver microsomes from phenobarbital sodium-pretreated rats. As determined by spectroscopic methods, these were identified as myrislignanometins A–G (2–8).Myrislignan (1), erythro-(1 R,2 S)-2-(4-allyl-2,6-dimethoxyphenoxyl)-1-(4-hydroxy-3-methoxyphenyl) propan-1-ol, is a major acyclic neolignan in seeds of Myristica fragrans. Studies have suggested that myrislignan may deter feeding activity, but little is known about its metabolism. We investigated the biotransformation of myrislignan by rat liver microsomes in vitro. Seven metabolites were produced by liver microsomes from rats pre-treated with sodium phenobarbital. These were identified, using spectroscopic methods, as myrislignanometins A–G (2–8), respectively.

Keywords: Myristica fragrans; Myristiceae; Biotransformation; Neolignan; Myrislignan; Erythro-(1; R; ,; 2; S; )-2-(4-allyl-2,6-dimethoxyphenoxyl)-1-(4-hydroxyl-3-methoxyphenyl) propan-1-ol; Myrislignanometins A–G

Diastereomeric stilbene glucoside dimers from the bark of Norway spruce ( Picea abies) by Sheng-Hong Li; Xue-Mei Niu; Stefan Zahn; Jonathan Gershenzon; Jennie Weston; Bernd Schneider (pp. 772-782).
Eight stilbene glucoside dimers containing either a dihydrofuran ring or a dihydro-1,4-dioxin moiety, designated as piceasides A–H, were isolated as four 1:1 mixtures of inseparable diastereomers. Their structures were determined by extensive spectroscopic means including 1D and 2D NMR spectra, and were supported by enzymatic hydrolysis and computational analysis.As part of a long-term study of the chemical defenses of Norway spruce ( Picea abies) against herbivores and pathogens, a phytochemical survey of the phenolics in the bark was carried out. Eight stilbene glucoside dimers, designated as piceasides A–H (1a4b), were isolated as four 1:1 mixtures of inseparable diastereomers. Their structures were determined by extensive spectroscopic means including 1D (1H and13C) and 2D NMR (1H–1H COSY, HSQC, HMBC, ROESY) spectra, and were supported by enzymatic hydrolysis and computational analysis.

Keywords: Norway spruce; Picea abies; Bark; Stilbene glucoside dimers; Piceasides A–H; Diastereomers

Metabolites from the endophytic fungus Phomopsis sp. PSU-D15 by Vatcharin Rukachaisirikul; Ubonta Sommart; Souwalak Phongpaichit; Jariya Sakayaroj; Kanyawim Kirtikara (pp. 783-787).
From the endophytic fungus Phomopsis sp. PSU-D15, three metabolites (13) were isolated together with three known compounds. Their structures were elucidated by spectroscopic methods and by comparison with the literature values. Compound1 exhibited moderate in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Ra with a MIC value of 6.25μg/ml.From the endophytic fungus Phomopsis sp. PSU-D15, three metabolites named as phomoenamide (1), phomonitroester (2) and deacetylphomoxanthone B (3), were isolated together with three known compounds, dicerandrol A (4), (1 S,2 S,4 S)- p-menthane-1,2,4-triol (5) and uridine. Their structures were elucidated by spectroscopic methods. Phomoenamide (1) exhibited moderate in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Ra.

Keywords: Phomopsis; sp.; Endophytic fungus; Secondary metabolites; Antimycobacterial activity

Neolignan glycosides from Symplocos caudata by Changhong Huo; Hong Liang; Yuying Zhao; Bin Wang; Qingying Zhang (pp. 788-795).
Four optical isomers of 7,9,9′-trihydroxy-3,3′-dimethoxy-8- O-4′-neolignan-4- O-β-d-glucopyranoside, a lignan lactone glycoside, and a phenylpropanoid glycoside were isolated from Symplocos caudataA phytochemical investigation of the roots of Symplocos caudata Wall (Symplocaceae) resulted in isolation and characterization of four optical isomers of a neolignan glycoside (14), a lignan lactone glycoside (5), a phenylpropanoid glycoside (6), as well as two known compounds (7,8). Their structures were elucidated as (7 S,8 S)- threo-7,9,9′-trihydroxy-3,3′-dimethoxy-8- O-4′-neolignan-4- O-β-d-glucopyranoside (1), (7 R,8 R)- threo-7,9,9′-trihydroxy-3,3′-dimethoxy-8- O-4′-neolignan-4- O-β-d-glucopyranoside (2), (7 R,8 S)- erythro-7,9,9′-trihydroxy-3,3′-dimethoxy-8- O-4′-neolignan-4- O-β-d-glucopyranoside (3), (7 S,8 R)- erythro-7,9,9′-trihydroxy-3,3′-dimethoxy-8- O-4′-neolignan-4- O-β-d-glucopyranoside (4), 8 R,8′ R-matairesinol-4- O-β-d-xylopyranosyl-(1→2)- O-β-d-glucopyranoside (5), 1- O-[β-d-xylopyranosyl-(1→6)- O-β-d-glucopyranosyl]-2,6-dimethoxy-4-propenyl-phenol (6), matairesinoside (7), and ( R)-1- O-(β-d-glucopyranosyl)-2-[2-methoxy-4-(ω-hydroxypropyl)-phenoxyl]-propan-3-ol (8) on the basis of spectroscopic data (1D and 2D NMR, MS and CD) and chemical evidence.

Keywords: Symplocos caudata; Symplocaceae; Lignan; Phenylpropanoid; Isomers of neolignan

Steroidal saponins from the roots of Asparagus racemosus by Patricia Y. Hayes; Aisyah H. Jahidin; Reg Lehmann; Kerry Penman; William Kitching; James J. De Voss (pp. 796-804).
Five steroidal saponins (shatavarins VI–X) and five known saponins have been isolated from the roots of Asparagus racemosus by RP-HPLC and then characterized by spectroscopic methods (1D and 2D NMR experiments) and mass spectrometry.Five steroidal saponins, shatavarins VI–X, together with five known saponins, shatavarin I (or asparoside B), shatavarin IV (or asparinin B), shatavarin V, immunoside and schidigerasaponin D5 (or asparanin A), have been isolated from the roots of Asparagus racemosus by RP-HPLC and characterized by spectroscopic (1D and 2D NMR experiments) and spectrometric (LCMS) methods.

Keywords: Saponins; Shatavarins; Asparagus racemosus; Asparagaceae; NMR

Monoterpene glycosides isolated from Fadogia agrestis by Regina Anero; Ana Díaz-Lanza; Evelyne Ollivier; Béatrice Baghdikian; Guy Balansard; Manuel Bernabé (pp. 805-811).
We report the isolation and structural elucidation of six monoterpene glycosides from Fadogia agrestis, a shrub used in traditional african medicine. The compounds are all derivatives of 2,6-dimethyl-2( E),6( Z)-octadiene-1,8-diol. In addition, three of them contain the acyl group B. The glycosides are derivatives of rhamnose and glucose.Six monoterpene glycosides were isolated from Fadogia agrestis. Their structures were elucidated using a combination of mass spectroscopy, 1D- and 2D-homo- and hetero-NMR spectroscopy and chemical analysis, and established as being derivatives of 2,6-dimethyl-2( E),6( Z)-octadiene-1,8-diol containing from two to four units of rhamnopyranose and, three of them, one or two additional units of glucopyranose. In three of the compounds an acyl group of 8-hydroxy-2,6-dimethyl-2( E),6( Z)-octadienoyl was found esterifying the O-2 position of one of the units of rhamnopyranose.

Keywords: Fadogia agrestis; Rubiaceae; Monoterpene glycosides

Ingenane diterpenoids from Euphorbia esula by Zhi-Qiang Lu; Min Yang; Jin-Qiang Zhang; Guang-Tong Chen; Hui-Lian Huang; Shu-Hong Guan; Chao Ma; Xuan Liu; De-An Guo (pp. 812-819).
Sixteen ingenane diterpenoids (116), together with five known compounds, were isolated from Euphorbia esula. Their structures were elucidated on the basis of spectroscopic studies and comparison with known related compounds. All the compounds were assayed for their inhibitory activity against human hela cervical cancer cell line.An extensive study of metabolites present in Euphorbia esula led to isolation of 16 ingenane diterpenoids116 together with the known ingenane derivative17 and four known cycloartane triterpenoids. Their structures were elucidated on the basis of spectroscopic studies and comparison with known related compounds. All the compounds were assayed for their inhibitory activity against human HeLa cervical cancer cell line.

Keywords: Euphorbia esula; Euphorbiaceae; Diterpenoids; Ingenane

An ellagitannin, n-butyl gallate, two aryltetralin lignans, and an unprecedented diterpene ester from Pelargonium reniforme by Klaus Peter Latté; Maki Kaloga; Andreas Schäfer; Herbert Kolodziej (pp. 820-826).
Four phenolic metabolites (pelargoniin E, n-buyl gallate, an aryltetralin lignan and, a unique diterpene ester, reniformin) together with five rarely reported compounds were isolated from Pelargonium reniforme.The structural diversity of the metabolic pool of Pelargonium reniforme was extended by the characterization of the1C4-glucose based ellagitannin pelargoniin E, gallic acid n-butyl ester, (−)-4,4′,9′-trihydroxy-3′,5′-dimethoxy-2,7′-cyclolignan 9- O-β-glucopyranoside and reniformin, a diterpene ester comprised of a diterpene acid with an uncommon –(CH2)2– bridging element linked to 2-(4-hydroxyphenyl)ethansulfonic acid. These metabolites were associated with the known (α,β)-3,4-di- O-galloyl-glucopyranoside, 4,6-dihydroxy-2β-glucopyranosyloxyacetophenone, 1- O-galloylglycerol, 6′- O-galloylsalidroside and (+)-isolariciresinol-9′- O-β-glucopyranoside. All structures were established on the basis of spectroscopic methods.

Keywords: Pelargonium sidoides; Ellagitannin; Pelargoniin E; Diterpene; Reniformin; Butyl gallate; Aryltetralin lignans

Antioxidant phenylpropanoid glycosides from the leaves of Wasabia japonica by Takahiro Hosoya; Young Sook Yun; Akira Kunugi (pp. 827-832).
From the MeOH extract of the leaves of wasabia japonica, seven phenylpropanoid glycosides (17) were isolated and determined based on spectroscopic data and chemical evidence along with eight known phenylpropanoids (815). The scavenging effect of compounds115 on superoxide anion radicals was investigated using an electron spin resonance method.From the MeOH extract of the leaves of W. japonica, seven phenylpropanoid gentiobiosides (17) were isolated along with eight known phenylpropanoids (815). Structures of17 were determined based on spectroscopic data and chemical evidence. The activity of compounds115 to scavenge superoxide anion radicals was investigated using an electron spin resonance (ESR) method.

Keywords: Wasabia japonica; Matsumura; Cruciferae; Electron spin resonance (ESR); Phenylpropanoid glycosides; Superoxide anion radicals

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