Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Laura Siles, Leonor Alegre, Verónica Tijero, Sergi Munné-Bosch Most angiosperms accumulate vitamin E in the form of tocopherols in seeds, exerting a protective antioxidant role. However, several palm trees principally accumulate tocotrienols, rather than tocopherols, in seeds, as it occurs in other monocots. To unravel the protective role of either tocopherols or tocotrienols against lipid peroxidation during seed germination in Chamaerops humilis var. humilis; seed viability, natural and induced germination capacity, seed water content, malondialdehyde levels (as an indicator of the extent of lipid peroxidation) and vitamin E levels (including both tocopherols and tocotrienols) were examined at various germination phases in a simulated, natural seed bank. At the very early stages of germination (operculum removal), malondialdehyde levels increased 2.8-fold, to decrease later up to 74%, thus indicating a transient lipid peroxidation at early stages of germination. Tocopherol levels were absent in quiescent seeds and did not increase during operculum removal, but increased later presumably dampening malondialdehyde accumulation. Thereafter, tocopherols continued increasing, while lipid peroxidation levels decreased. By contrast, tocotrienols levels remained constant or even decreased as germination progressed, showing no correlation with lipid peroxidation levels. We hypothesize that despite their high tocotrienol content, seeds synthesize tocopherols during germination to protect lipids from peroxidation events.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Christina Braunberger, Martin Zehl, Jürgen Conrad, Christoph Wawrosch, Jaqueline Strohbach, Uwe Beifuss, Liselotte Krenn The botanical classification of the huge genus Drosera remains controversial since long. In the present study, the pattern of major phenolic compounds in ten Drosera species belonging to seven different subgenera and/or sections of the genus was investigated for chemotaxonomic allocation. The composition of flavonoids and ellagic acid derivatives in Drosera adelae, Drosera burmannii, Drosera dielsiana, Drosera hilaris, Drosera montana, Drosera petiolaris, and Drosera pygmaea was elucidated for the first time. The scarce data on these compounds in Drosera binata, Drosera aliciae, and Drosera spatulata were complemented significantly. Detailed LC–DAD–MS, LC–NMR, and offline 1D and 2D NMR analyses resulted in the unambiguous identification of around 40 different substances, three of them (8-hydroxy-luteolin-8-O-arabinopyranoside, tricetin-7-O-xylopyranoside and 8-hydroxytricetin-8-O-arabinopyranoside) being natural products described for the first time. The distribution of the compounds characterized underlines their potential to serve as chemotaxonomic markers in this genus.
Publication date: September 2015 Source:Phytochemistry, Volume 117 Author(s): Hafeeza Khatoon, Johanna Mansfeld, Angelika Schierhorn, Renate Ulbrich-Hofmann Phospholipase D (PLD; E.C. 126.96.36.199) 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 92.0kDa, pI 5.39, maximum activity at pH 5.5–6.0 and Ca2+ ion concentrations ?60mM), allowed the assignment of this enzyme to the class of ?-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?molmin?1mg?1 and 1.84±0.17mM, respectively. Phosphate analogs such as NaAlF4 and Na3VO4 displayed strong inhibition of the enzyme. Phosphatidylinositol 4,5-bisphosphate had a strong activating effect at 2–10mM 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.
Phospholipase D from Indian mustard seeds was purified to homogeneity. Amino acid sequence data and enzymological properties suggest its assignment to ?-type phospholipases D
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Caroline Müller, Joop van Loon, Sara Ruschioni, Gina Rosalinda De Nicola, Carl Erik Olsen, Renato Iori, Niels Agerbirk Isothiocyanates (ITCs), released from Brassicales plants after hydrolysis of glucosinolates, are known for their negative effects on herbivores but mechanisms have been elusive. The ITCs are initially present in dissolved form at the site of herbivore feeding, but volatile ITCs may subsequently enter the gas phase and all ITCs may react with matrix components. Deterrence to herbivores resulting from topically applied volatile ITCs in artificial feeding assays may hence lead to ambiguous conclusions. In the present study, the non-volatile ITC moringin (4-(?-l-rhamnopyranosyloxy)benzyl ITC) and its glucosinolate precursor glucomoringin were examined for effects on behaviour and taste physiology of specialist insect herbivores of Brassicales. In feeding bioassays, glucomoringin was not deterrent to larvae of Pieris napi (Lepidoptera: Pieridae) and Athalia rosae (Hymenoptera: Tenthredinidae), which are adapted to glucosinolates. Glucomoringin stimulated feeding of larvae of the related Pieris brassicae (Lepidoptera: Pieridae) and also elicited electrophysiological activity from a glucosinolate-sensitive gustatory neuron in the lateral maxillary taste sensilla. In contrast, the ITC moringin was deterrent to P. napi and P. brassicae at high levels and to A. rosae at both high and low levels when topically applied to cabbage leaf discs (either 12, 120 or 1200nmol moringin per leaf disc of 1cm diameter). Survival of A. rosae was also significantly reduced when larvae were kept on leaves treated with moringin for several days. Furthermore, moringin elicited electrophysiological activity in a deterrent-sensitive neuron in the medial maxillary taste sensillum of P. brassicae, providing a sensory mechanism for the deterrence and the first known ITC taste response of an insect. In simulated feeding assays, recovery of moringin was high, in accordance with its non-volatile nature. Our results demonstrate taste-mediated deterrence of a non-volatile, natural ITC to glucosinolate-adapted insects.
The non-volatile isothiocyanate moringin deterred larvae of the sawfly Athalia rosae and two Pieris species, and elicited response from a deterrent taste neuron in P. brassicae. Recovery of moringin applied to leaf discs for feeding assays was high.
Publication date: Available online 8 October 2015 Source:Phytochemistry Author(s): Hua-Wei Lv, Jian-Guang Luo, Meng-Di Zhu, Hui-Jun Zhao, Ling-Yi Kong Seven neo-clerodane diterpenes, teufruintins A–G (1–7), together with eight known compounds (8–15) were isolated from the CHCl3-soluble fraction of the aerial parts of Teucrium fruticans cultivated in China. The chemical structures of the isolated compounds were elucidated using different spectroscopic methods. All of the isolated diterpenes were evaluated for their cytotoxic activities on three human cancer cell lines, and for their ability to inhibit LPS-induced nitric oxide production in RAW 264.7 macrophages. None of the compounds displayed cytotoxic activities on the cancer cell lines, and only 15 showed weak NO inhibitory activity.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Tahira Fatima, Vigya Kesari, Ian Watt, David Wishart, James F. Todd, William R. Schroeder, Gopinadhan Paliyath, Priti Krishna In this study, phenolic compounds were analyzed in developing berries of four Canadian grown sea buckthorn (Hippophae rhamnoides L.) cultivars (‘RC-4’, ‘E6590’, ‘Chuyskaya’ and ‘Golden Rain’) and in leaves of two of these cultivars. Among phenolic acids, p-coumaric acid was the highest in berries, while gallic acid was predominant in leaves. In the flavonoid class of compounds, myricetin/rutin, kaempferol, quercetin and isorhamnetin were detected in berries and leaves. Berries of the ‘RC-4’ cultivar had approximately ?2-fold higher levels of myricetin and quercetin at 17.5mg and 17.2mg/100g FW, respectively, than the other cultivars. The flavonoid content in leaves was considerably more than in berries with rutin and quercetin levels up to 135mg and 105mg/100g FW, respectively. Orthologs of 15 flavonoid biosynthesis pathway genes were identified within the transcriptome of sea buckthorn mature seeds. Semi-quantitative RT-PCR analysis of these genes in developing berries indicated relatively higher expression of genes such as CHS, F3?H, DFR and LDOX in the ‘RC-4’ cultivar than in the ‘Chuyskaya’ cultivar. Vitamin C levels in ripened berries of the Canadian cultivars were on the high end of the concentration range reported for most other sea buckthorn cultivars. Orthologs of genes involved in vitamins C and E biosynthesis were also identified, expanding the genomic resources for this nutritionally important plant.
Publication date: Available online 8 September 2015 Source:Phytochemistry Author(s): Kyo Bin Kang, Gao Ming, Geum Jin Kim, Thi-Kim-Quy Ha, Hyukjae Choi, Won Keun Oh, Sang Hyun Sung Five Ib-type cyclopeptide alkaloids, jubanines F–J (1–5), and three known compounds, nummularine B (6), daechuine-S3 (7), and mucronine K (8) were isolated from the roots of Ziziphus jujuba. Their structures were fully characterized by spectroscopic analyses in combination with chemical derivatization. Compounds 1–3, and 6 were evaluated for their antiviral activity against the porcine epidemic diarrhea virus (PEDV). Compounds 2, 3, and 6 showed potent inhibitory effects on PEDV replication.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Marie-Sophie Louvet, Gilbert Gault, Sébastien Lefebvre, Florence Popowycz, Manon Boulven, Stéphane Besse, Etienne Benoit, Virginie Lattard, Denis Grancher Two distinguishable chemotypes of Ferula communis have been described: the ‘nonpoisonous’ chemotype, containing as main constituents the daucane esters; and the ‘poisonous’ chemotype containing prenylated coumarins, such as ferulenol and ferprenin. Ferulenol and ferprenin are 4-oxygenated molecules such as dicoumarol and warfarin, the first developed antivitamin K molecules. Antivitamin K molecules specifically inhibit VKORC1, an enzyme essential for recycling vitamin K. This latest is involved in the activation of clotting factors II, VII, IX, X. The inhibiting effect of ferulenol on VKORC1 was shown in rat, but not for species exposed to F. communis while in vivo studies suggest differences between animal susceptibility to ferulenol. The inhibiting effect of ferprenin on VKORC1 was never demonstrated. The aim of this study was to compare the inhibiting effect of both compounds on VKORC1 of different species exposed to F. communis. Vitamin K epoxide activity was evaluated for each species from liver microsomes and inhibiting effect of ferulenol and ferprenin was characterized. Ferulenol and ferprenin were shown to be able to inhibit VKORC1 from all analyzed species. Nevertheless, susceptibility to ferulenol and ferprenin presented differences between species, suggesting a different susceptibility to ‘poisonous’ chemotypes of F. communis.
Publication date: Available online 8 October 2015 Source:Phytochemistry Author(s): Qian Hao, Yoshinori Saito, Yosuke Matsuo, Hai-Zhou Li, Takashi Tanaka A detailed chemical investigation of Chinese Dragon’s Blood, which is a traditional medicine produced form the red resin of Dracaena cochinchinensis, yielded two chalcane–stilbene conjugates, named cochinchinenenes G and H, together with 25 known compounds. The structures of these compounds were determined by spectroscopic examination. HPLC analysis of the resin indicated that the major constituents were a complex mixture of oligomeric polyphenols, which were detected as a broad hump on the base line of a HPLC chromatogram. 13C NMR analysis indicated that the oligomers were mainly composed of oxygenated chalcane units. This suggestion was supported by the results of a thiol degradation experiment with mercaptoethanol, which yielded a thioether of 4-[(4-hydroxyphenyl)propyl]-3-methoxyphenol. Furthermore, methylation followed by electrospray ionization mass spectroscopic analysis of the resulting fractions established the presence of at least one heptamer of chalcane units.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): B.L. Mikkelsen, C.E. Olsen, M.F. Lyngkjær Plants produce secondary metabolites promoting adaptation to changes in the environment and challenges by pathogenic microorganisms. A future climate with increased temperature and CO2 and ozone levels will likely alter the chemical composition of plants and thereby plant–pathogen interactions. To investigate this, barley was grown at elevated CO2, temperature and ozone levels as single factors or in combination resembling future climatic conditions. Increased basal resistance to the powdery mildew fungus was observed when barley was grown under elevated CO2, temperature and ozone as single factors. However, this effect was neutralized in the combination treatments. Twenty-five secondary metabolites were putatively identified in healthy and diseased barley leaves, including phenylpropanoids, phenolamides and hydroxynitrile glucosides. Accumulation of the compounds was affected by the climatic growth conditions. Especially elevated temperature, but also ozone, showed a strong impact on accumulation of many compounds, suggesting that these metabolites play a role in adaptation to unfavorable growth conditions. Many compounds were found to increase in powdery mildew diseased leaves, in correlation with a strong and specific influence of the climatic growth conditions. The observed disease phenotypes could not be explained by accumulation of single compounds. However, decreased accumulation of the powdery mildew associated defense compound p-coumaroylhydroxyagmatine could be implicated in the increased disease susceptibility observed when barley was grown under combination of elevated CO2, temperature and ozone. The accumulation pattern of the compounds in both healthy and diseased leaves from barley grown in the combination treatments could not be deduced from the individual single factor treatments. This highlights the complex role and regulation of secondary metabolites in plants’ adaptation to unfavorable growth conditions.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Kai-Long Ji, Ping Zhang, Xiao-Nian Li, Juan Guo, Hua-Bin Hu, Chun-Fen Xiao, Xiang-Qun Xie, You-Kai Xu Ten cedrelone limonoids were isolated from the leaves of Trichilia americana. These compounds include americanolides A–D (1–4), 1,2-dihydrodeacetylhirtin (5), 1?-hydroxy-1,2-dihydrodeacetylhirtin (6), 1?-hydroxy-1,2-dihydrohirtin (7), 1?-methoxy-1,2-dihydrodeacetylhirtin (8), 11?-hydroxy-12?-propanoyloxycedrelone (9), and 1?,11?-dihydroxy-1,2-dihydrocedrelone (10), as well as two previously reported compounds, deacetylhirtin (11) and hirtin (12). Their structures were characterized on the basis of spectroscopic studies, and the assignment of the absolute configuration of americanolide A (1) was supported by single-crystal X-ray diffraction studies. The cytotoxic activities of all isolated compounds were also evaluated against five human tumour cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480) using an MTS assay. Compounds 11 and 12 showed significant cytotoxicity with IC50 values ranging from 0.1 to 0.5?M, and compounds 5, 6, 7, 8, 9, and 10 exhibited potent or selective cytotoxic activity with IC50 values ranging from 1.0 to 39.6?M.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Han Wei, Ya-ming Xu, Patricia Espinosa-Artiles, Manping X. Liu, Jiang-Guang Luo, Jana M. U’Ren, A. Elizabeth Arnold, A.A. Leslie Gunatilaka Oxygenated guaiane-type sesquiterpenes, xylaguaianols A?D (1–4), an iso-cadinane-type sesquiterpene isocadinanol A (5), and an ?-pyrone 9-hydroxyxylarone (6), together with five known sesquiterpenes (7–11), and four known cytochalasins (12–15) were isolated from a culture broth of Xylaria sp. NC1214, a fungal endophyte of the moss Hypnum sp. The structures of all compounds were elucidated by the analysis of their spectroscopic data and relative configurations of 1–5 were determined with the help of NMR NOESY experiments. Cytochalasins C (12), D (13), and Q (14) were investigated for their cytotoxic activity against five tumor cell lines. Cytochalasin D showed significant cytotoxicity against all five cell lines, with IC50s ranging from 0.22 to 1.44?M, whereas cytochalasins C and Q exhibited moderate, but selective cytotoxicity.
Publication date: Available online 5 October 2015 Source:Phytochemistry Author(s): Marcio V. Ramos, Raquel S.B. de Oliveira, Humberto M. Pereira, Frederico B.M.B. Moreno, Marina D.P. Lobo, Luciana M. Rebelo, José Brandão-Neto, Jeanlex S. de Sousa, Ana C.O. Monteiro-Moreira, Cléverson D.T. Freitas, Thalles Barbosa Grangeiro CpOsm is an antifungal osmotin/thaumatin-like protein purified from the latex of Calotropis procera. The protein is relatively thermostable and retains its antifungal activity over a wide pH range; therefore, it may be useful in the development of new antifungal drugs or transgenic crops with enhanced resistance to phytopathogenic fungi. To gain further insight into the mechanism of action of CpOsm, its three-dimensional structure was determined, and the effects of the protein on Fusarium solani spores were investigated by atomic force microscopy (AFM). The atomic structure of CpOsm was solved at a resolution of 1.61Å, and it contained 205 amino acid residues and 192 water molecules, with a final R-factor of 18.12% and an Rfree of 21.59%. The CpOsm structure belongs to the thaumatin superfamily fold and is characterized by three domains stabilized by eight disulfide bonds and a prominent charged cleft, which runs the length of the front side of the molecule. Similarly to other antifungal thaumatin-like proteins, the cleft of CpOsm is predominantly acidic. AFM images of F. solani spores treated with CpOsm resulted in striking morphological changes being induced by the protein. Spores treated with CpOsm were wrinkled, and the volume of these cells was reduced by approximately 80%. Treated cells were covered by a shell of CpOsm molecules, and the leakage of cytoplasmic content from these cells was also observed. Based on the structural features of CpOsm and the effects that the protein produces on F. solani spores, a possible mechanism of action is suggested and discussed.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Mike Pollard, Danielle Delamarter, Tina M. Martin, Yair Shachar-Hill Studies on the metabolism of lipids in seeds frequently use radiolabeled acetate and glycerol supplied to excised developing seeds to track the biosynthesis of acyl and lipid head groups, respectively. Such experiments are generally restricted to shorter time periods and the results may not quantitatively reflect in planta rates. These limitations can be removed by using cultured embryos, provided they mimic growth and lipid deposition observed for embryos in planta. Mid-maturation embryos from Camelina sativa were cultured in vitro to assess the use of sufficient acetate or glycerol concentrations and labeling periods for stable isotope labeling and mass spectrometric detection. Maximum incorporation of exogenous acetate into fatty acids occurred at 1mM and above. This provides about 5% of the total carbon flux entering fatty acids, enough for 13C isotopomer analysis while maintaining normal biosynthetic rates for over 24h. Labeling analysis indicates that acetate reports lipid metabolism uniformly across the embryo. At higher acetate concentrations with longer incubations, the rate of fatty acid synthesis is reduced and the composition of newly synthesized fatty acids changes. While the mole fractions of oleate that undergo ?12-desaturation or elongation are independent of biosynthetic flux, ?15-desaturation shows a bimodal dependence. These observations are consistent with changes occurring in planta over seed development. Incorporation rates of the glyceryl moiety into lipids saturates at about 0.5mM exogenous glycerol. At saturation, the exogenous glycerol almost completely replaces the endogenous supply of glycerol-3-phosphate without affecting net lipid accumulation or fatty acid composition. It is concluded that acetate and glycerol labeling of cultured C. sativa embryos can provide an accurate representation of lipid metabolism in embryos in vivo, and that in Camelina embryos glycerol-3-phosphate levels do not co-limit triacylglycerol synthesis.
Publication date: Available online 24 September 2015 Source:Phytochemistry Author(s): Otmar Spring, Jens Pfannstiel, Iris Klaiber, Jürgen Conrad, Uwe Beifuß, Lysanne Apel, Anna-Katharina Aschenbrenner, Reinhard Zipper Uniseriate linear glandular trichomes occur on stems, leaves and flowering parts of Helianthus species and related taxa. Their metabolic activity and biological function are still poorly understood. A phytochemical study documented the accumulation of bisabolene type sesquiterpenes and flavonoids as the major constituents of the non-volatile metabolome of linear glandular trichomes in the common sunflower, Helianthus annuus. Besides known sesquiterpenes of the glandulone, helibisabonol and heliannuol type, four previously undescribed sesquiterpenes named glandulone D, E, F and helibisabonol C were identified by spectroscopic analysis. In addition, four known nevadensin type flavonoids varying in O-methoxy substitutions were found. None of them has previously been reported from Helianthus annuus.
HPLC analysis of the metabolite profile of sunflower trichomes afforded 4 previously undescribed sesquiterpenes of the helibisabonol- and glandulone-type, and proved the presence of various nevadensin derived flavonoids in the same compartment.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Ewa Latkowska, Beata Bober, Ewelina Chrapusta, Michal Adamski, Ariel Kaminski, Jan Bialczyk Lichen species typically have a characteristic profile of secondary metabolites. Dense populations of Hypogymnia physodes growing frequently as epiphytes on tree branches have harmful effects on the host, likely due to their secondary compounds, which were undetected in tree tissues until now. The aim of the present study was to re-characterise the suite of secondary metabolites of H. physodes thalli and to estimate their translocation into spruce (Picea abies) bark. Thallus and bark extracts were compared using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). The compounds were identified based on their UV, MS and MS/MS spectra as well as retention factors of their TLC analysis. In addition to the previously described secondary metabolites (protocetraric, physodalic, 3-hydroxyphysodic, physodic, and 2?-O-methylphysodic acids, atranorin and chloroatranorin) of H. physodes, further three were identified in its thalli: conphysodalic, 4-O-methylphysodic and ?-alectoronic acids. Fragmentation patterns from the negative ionisation of each compound were proposed, some of which were described for the first time. Among all of the detected lichen substances, a few, e.g., physodalic, 3-hydroxyphysodic, physodic acids and atranorin, were present in the bark of spruce branches that were abundantly colonised by lichen. The newly identified compounds of H. physodes thalli may belong to its constant or accessory secondary metabolites. These compounds may be useful in the chemotaxonomic classification of this species. The presence of some lichen substances in spruce bark confirmed their ability to penetrate host tissues. These data suggest that H. physodes compounds may cause long-term effects on spruces in nature.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): M. Soledade C. Pedras, Mahla Alavi, Q. Huy To The metabolites produced in leaves of the crucifers winter cress (Barbarea vulgaris) and upland cress (Barbarea verna) abiotically elicited were investigated and their chemical structures were elucidated by analyses of spectroscopic data and confirmed by syntheses. Nasturlexins C and D and their sulfoxides are cruciferous phytoalexins displaying antifungal activity against the crucifer pathogens Alternaria brassicicola, Leptosphaeria maculans and Sclerotinia sclerotiorum. The biosynthesis of these metabolites is proposed based on pathways of cruciferous indolyl phytoalexins. This work indicates that B. vulgaris and B. verna have great potential as sources of defense pathways transferable to agriculturally important crops within the Brassica species.
Publication date: September 2015 Source:Phytochemistry, Volume 117 Author(s): Liuyi Dang, Els J.M. Van Damme Plants 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.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Helga Pankoke, Ingo Höpfner, Agnieszka Matuszak, Wolfram Beyschlag, Caroline Müller Plants are sessile organisms that suffer from a multitude of challenges such as abiotic stress or the interactions with competitors, antagonists and symbionts, which influence their performance as well as their eco-physiological and biochemical responses in complex ways. In particular, the combination of different stressors and their impact on plant biomass production and the plant’s ability to metabolically adjust to these challenges are less well understood. To study the effects of mineral nitrogen (N) availability, interspecific competition and the association with arbuscular mycorrhizal fungi (AMF) on biomass production, biomass allocation patterns (root/shoot ratio, specific leaf area) and metabolic responses, we chose the model organism Plantago lanceolata L. (Plantaginaceae). Plants were grown in a full factorial experiment. Biomass production and its allocation patterns were assessed at harvest, and the influence of the different treatments and their interactions on the plant metabolome were analysed using a metabolic fingerprinting approach with ultra-high performance liquid chromatography coupled with time-of-flight-mass spectrometry. Limited supply of mineral N caused the most pronounced changes with respect to plant biomass and biomass allocation patterns, and altered the concentrations of more than one third of the polar plant metabolome. Competition also impaired plant biomass production, yet affected the plant metabolome to a much lesser extent than limited mineral N supply. The interaction of competition and limited mineral N supply often caused additive changes on several traits. The association with AMF did not enhance biomass production, but altered biomass allocation patterns such as the root/shoot ratio and the specific leaf area. Interestingly, we did not find significant changes in the plant metabolome caused by AMF. A targeted analysis revealed that only limited mineral N supply reduced the concentrations of one of the main target defence compounds of P. lanceolata, the iridoid glycoside catalpol. In general, the interaction of competition and limited mineral N supply led to additive changes, while the association with AMF in any case alleviated the observed stress responses. Our results show that the joint analysis of biomass/allocation patterns and metabolic traits allows a more comprehensive interpretation of plant responses to different biotic and abiotic challenges; specifically, when multiple stresses interact.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Owen M. McDougal, Peter B. Heenan, Peter Jaksons, Catherine E. Sansom, Bruce M. Smallfield, Nigel B. Perry, John W. van Klink Alkaloid contents of leaf and seed samples of eight species of Sophora native to New Zealand, plus Sophora cassioides from Chile are reported. Fifty-six leaf and forty-two seed samples were analysed for alkaloid content by proton nuclear magnetic resonance spectroscopy, which showed major alkaloids as cytisine, N-methyl cytisine and matrine. GC analyses quantified these and identified further alkaloid components. The alkaloids identified were cytisine, sparteine, and matrine-types common to Sophora from other regions of the world. Cytisine, N-methyl cytisine, and matrine were generally the most abundant alkaloids across all species with seeds containing the highest concentrations of alkaloids. However, there was no clear taxonomic grouping based on alkaloid composition. A quantitative analysis of various parts of two Sophora microphylla trees showed that the seeds were the richest source of alkaloids (total 0.4–0.5% DM), followed by leaf and twig (0.1–0.3%) and then bark (0.04–0.06%), with only low amounts (<0.02%) found in the roots. This study represents the most comprehensive phytochemical investigation of New Zealand Sophora species to date and presents data for three species of Sophora for which no prior chemistry has been reported.
Publication date: Available online 8 October 2015 Source:Phytochemistry Author(s): Brigitte Lukas, Corinna Schmiderer, Johannes Novak This investigation focused on the qualitative and quantitative composition of essential oil compounds of European Origanum vulgare. Extracts of 502 individual O. vulgare plants from 17 countries and 51 populations were analyzed via GC. Extracts of 49 plants of 5 populations of Israeli Origanum syriacum and 30 plants from 3 populations of Turkish Origanum onites were included to exemplify essential oil characteristics of ‘high-quality’ oregano. The content of essential oil compounds of European O. vulgare ranged between 0.03% and 4.6%. The monoterpenes were primarily made up of sabinene, myrcene, p-cymene, 1,8-cineole, ?-ocimene, ?-terpinene, sabinene hydrate, linalool, ?-terpineol, carvacrol methyl ether, linalyl acetate, thymol and carvacrol. Among the sesquiterpenes ?-caryophyllene, germacrene D, germacrene D-4-ol, spathulenol, caryophyllene oxide and oplopanone were often present in higher amounts. According to the proportions of cymyl-compounds, sabinyl-compounds and the acyclic linalool/linalyl acetate three different main monoterpene chemotypes were defined. The cymyl- and the acyclic pathway were usually active in plants from the Mediterranean climate whereas an active sabinyl-pathway was a characteristic of plants from the Continental climate.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Zakaria Cheikh-Ali, Kyriaki Glynou, Tahir Ali, Sebastian Ploch, Marcel Kaiser, Marco Thines, Helge B. Bode, Jose G. Maciá-Vicente Members of the fungal genus Exophiala are common saprobes in soil and water environments, opportunistic pathogens of animals, or endophytes in plant roots. Their ecological versatility could imply a capacity to produce diverse secondary metabolites, but only a few studies have aimed at characterizing their chemical profiles. Here, we assessed the secondary metabolites produced by five Exophiala sp. strains of a particular phylotype, isolated from roots of Microthlaspi perfoliatum growing in different European localities. Exophillic acid and two previously undescribed compounds were isolated from these strains, and their structures were elucidated by spectroscopic methods using MS, 1D and 2D NMR. Bioassays revealed a weak activity of these compounds against disease-causing protozoa and mammalian cells. In addition, 18 related structures were identified by UPLC/MS based on comparisons with the isolated structures. Three Exophiala strains produced derivatives containing a ?-d-glucopyranoside moiety, and their colony morphology was distinct from the other two strains, which produced derivatives lacking ?-d-glucopyranoside. Whether the chemical/morphological strain types represent variants of the same genotype or independent genetic populations within Exophiala remains to be evaluated.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Rui-Xue Xu, Yu Zhao, Shuai Gao, Yu-Ying Zhang, Dan-Dan Li, Hong-Xiang Lou, Ai-Xia Cheng Caffeoyl CoA O-methyltransferases (CCoAOMTs), known to be involved in phenylpropanoid metabolism and lignin synthesis, have been characterized from several higher plant species, which also harbor CCoAOMT-like enzymes responsible for methylation of a variety of flavonoids, anthocyanins, coumarins and phenylpropanoids. Here, a gene encoding a CCoAOMT (PaOMT1) was isolated from a sequenced cDNA library of the liverwort species Plagiochasma appendiculatum, a species belonging to the Family Aytoniaceae. The full-length cDNA sequence of PaOMT1 contains 909bp, and is predicted to encode a protein with 302 amino acids. The gene products were 40–50% identical to CCoAOMT sequences of other plants. Experiments based on recombinant PaOMT1 showed that the enzyme was able to methylate phenylpropanoids, flavonoids and coumarins, with a preference for the flavonoid quercetin (19). Although the substrate selectivity and biochemical feature of PaOMT1 is similar to CCoAOMT-like enzymes, the sequence alignment results indicated PaOMT1 is closer to true CCoAOMT enzymes. A phylogenetic analysis indicated that PaOMT1 is intermediate between true CCoAOMTs and CCoAOMT-like enzymes. The transient expression of a PaOMT1-GFP fusion in tobacco demonstrated that PaOMT1 is directed to the plastids. PaOMT1 may represent an ancestral form of higher plant true CCoAOMT and CCoAOMT-like enzymes. This is the first time an O-methyltransferase was characterized in liverworts.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Fa-Wu Dong, Zhi-Kun Wu, Liu Yang, Chen-Ting Zi, Dan Yang, Rui-Jing Ma, Zhen-Hua Liu, Huai-Rong Luo, Jun Zhou, Jiang-Miao Hu Twenty-one compounds (nine iridoids and twelve sesquiterpenoids), including ten previously unknown (five iridoids and five sesquiterpenoids) were isolated from whole dried material of Valeriana stenoptera. Structures were established on the basis of extensive spectroscopic analysis and the relative stereochemistry of 13-hydroxypatchoulol A was further confirmed by X-ray crystallographic data. All isolates were evaluated for their effects on nerve growth factor (NGF)-mediated neurite outgrowth in pheochromocytoma (PC12) cells and seven compounds showed potent promoting effects.
Phytochemical research of Valeriana stenoptera resulted in isolation of many iridoids and sesquiterpenoids. Seven of these compounds showed promoting effects on NGF-induced neurite outgrowth in PC12 cells at a concentration of 10?M.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Anna Kim, Janggyoo Choi, Khin Myo Htwe, Young-Won Chin, Jinwoong Kim, Kee Dong Yoon Phytochemical investigations of the aerial parts of Acacia pennata (Mimosaceae) from Myanmar led to the isolation of five flavonoid glycosides and six known compounds. The new compounds were identified as (2R,3S)-3,5,7-trihdyroxyflavan-3-O-?-l-rhamnopyranoside, (2S)-5,7-dihydroxyflavan-7-O-?-d-glucopyranoside-(4??8)-epiafzelechin-3-O-gallate, (2R)-4?,7-dihydroxyflavan-(4??8)-(2R,3S)-3,5,7-trihdyroxyflavan-3?-O-?-l-rhamnopyranoside, 5,7-dihydroxyflavone 6-C-?-boivinopyranosyl-7-O-?-d-glucopyranoside, and 5,7-dihydroxyflavone 7-O-?-d-glucopyranosyl-8-C-?-boivinopyranoside based on interpretation of spectroscopic data.
Five flavonoid glycoside (1–5) together with six known compounds were isolated from the aerial parts of Acacia pennata in Myamar. Their structures were determined by 1D, 2D NMR, UV, MS and CD data.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Eric Kuhnert, Frank Surup, Jennifer Herrmann, Volker Huch, Rolf Müller, Marc Stadler Our screening efforts for new natural products with interesting bioactivity have revealed the neotropical ascomycete Hypoxylon rickii as a prolific source. We isolated five secondary metabolites with a p-terphenyl backbone from the mycelial extract of a fermentation of this fungus in 70l scale by using RP-HPLC, which were named rickenyls A–E (1–5). Their structures were elucidated by X-ray crystallography and NMR spectroscopy, complemented by HRESIMS. Two of the compounds contained a quinone core structure in ortho (2) and para-position (5), respectively. We obtained 2 spontaneously and by lead tetraacetate oxidation from 1. All compounds were screened for antimicrobial, antioxidative and cytotoxic activities. Rickenyl A (1) exhibited strong antioxidative effects and moderate cytotoxic activity against various cancer cell lines.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Xiao Chun Li, Yuan Yang Liao, David W.M. Leung, Hai Yan Wang, Bai Ling Chen, Xin Xiang Peng, E.E. Liu The biochemical and enzymatic properties of four highly similar rice oxalate oxidase proteins (OsOxO1–4) were compared after their purification from the leaves of transgenic plants each overexpressing the respective OsOxO1–4 genes. Although alignment of their amino acid sequences has revealed divergence mainly in the signal peptides and they catalyze the same enzymic (oxalate oxidase) reaction, divergence in apparent molecular mass, Km, optimum pH, stability and responses to inhibitors and activators was uncovered by biochemical characterization of the purified OsOxO1–4 proteins. The apparent molecular mass of oligomer OsOxO1 was found to be similar to that of OsOxO3 but lower than the other two. The molecular mass of the subunit of OsOxO1 was lower than that of OsOxO3. The Km value of OsOxO3 was higher than the other three which had similar Km. OsOxO1 and OsOxO4 possessed peak activity at pH 8.5 which was close to that at the optimum pH 4.0. The activity of OsOxO2 at pH 8.5 was only 65% of that at its optimum pH 3.5, while the activity of OsOxO3 did not vary much at pH 6–9 and was also much lower than that at its optimum pH 3. OsOxO2 and OsOxO3 still maintained all their activities after being heated at 70°C for 1h while OsOxO1 and OsOxO4 lost about 30% of their activities. Pyruvate and oxaloacetic acid inhibited the activity of OsOxO3 more strongly than the other three. Interestingly, glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-biphosphate related to photosynthetic assimilation of triose phosphate greatly increased the activities of OsOxO3 and OsOxO4. In addition to the differences in the biochemical properties of the four OsOxO proteins, an intriguing finding is that the purified OsOxO1–4 exhibited substrate inhibition, which is a typical of the classical Michaelis–Menten enzyme kinetics exhibited by a majority of other enzymes.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Masahiko Isaka, Arunrat Yangchum, Sumalee Supothina, Thitiya Boonpratuang, Rattaket Choeyklin, Palangpon Kongsaeree, Samran Prabpai Twelve aromadendrane sesquiterpenoids, inonotins A–L, and a previously unknown cyclofarnesane, i.e., inonofarnesane, together with two known compounds, were isolated from cultures of the wood-rotting basidiomycete Inonotus sp. BCC 23706. Inonotin I is identical to a previously reported compound with an incorrect structure. Structures of the compounds were elucidated by spectroscopic analysis and X-ray crystallography. The absolute configurations of inonotin D and inonofarnesane were determined by application of the modified Mosher’s method.
Publication date: Available online 28 September 2015 Source:Phytochemistry Author(s): Aline Lamien-Meda, Martin Kiendrebeogo, Moussa Compaoré, Roland N.T. Meda, Markus Bacher, Karin Koenig, Thomas Pacher, Hans-Peter Fuehrer, Harald Noedl, Merlin Willcox, Johannes Novak The present study focuses on development of phytochemical methods for quality assessment of two West-African Cochlospermum species (Cochlospermum planchonii and Cochlospermum tinctorium) traditionally used for malaria treatment in Burkina Faso. Antimalarial activity of preparations from dried rhizomes (decoction) was tested against the chloroquine-sensitive Plasmodium strain 3D7 using the histidine-rich protein II (HRP2) drug susceptibility assay and compared with extract preparations using organic solvents of different polarity. Two main apocarotenoids were isolated from rhizomes of C. planchonii and unambiguously identified as dihydrocochloxanthine and cochloxanthine by spectroscopic methods. Comparative HPLC analyses of thirty-nine (39) samples from markets and from collections in natural habitats of both species showed a high variability in the accumulation of cochloxanthines and related carotenoids which were proven to be characteristic for rhizomes of both species and generally absent in leaves. Furthermore, content of total phenolics and antioxidant activities (DPPH and FRAP) as well as haemolytic activity of various extracts was tested. The HPLC method presented here was validated and provides a good separation of both compounds including 10 minor carotenoids. Extracts from both species and pure cochloxanthine offered pronounced antioxidant activities and weak haemolytic activity while, in contrast, dihydrocochloxanthine had a strong haemolytic effect at the highest concentration analysed. However, cochloxanthine as well as dihydrocochloxanthine showed erythroprotective effects against the haemolytic activity of the reference saponin. Moderate antiplasmodial activity between 16 and 63?g/ml were observed with all tested extracts, and lower IC50 values were obtained with pure dihydrocochloxanthine (IC50=6.9?g/ml), cochloxanthine (IC50=6.8?g/ml), the DCM fraction (IC50=2.4?g/ml) and the ethyl acetate fraction (IC50=11.5?g/ml) derived from a methanolic extract of C. planchonii.This study shows a major variability of carotenoid content and antiplasmodial activity of both C. planchonii and C. tinctorium. The high haemolytic activity of dihydrocochloxanthine (at 100?g/ml) should be considered as a selection criterion for choosing species phenotypes for treatment.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Niels Agerbirk, Gina Rosalinda De Nicola, Carl Erik Olsen, Caroline Müller, Renato Iori Isothiocyanates form adducts with a multitude of biomolecules, and these adducts need analytical methods. Likewise, analytical methods for hydrophilic isothiocyanates are needed. We considered reaction with ammonia to form thiourea derivatives. The hydrophilic, glycosylated isothiocyanate moringin, 4-(?-l-rhamnopyranosyloxy)benzyl isothiocyanate, was efficiently derivatized to the thiourea derivative by incubation with ammonia. The hydrophobic benzyl isothiocyanate was also efficiently derivatized to the thiourea derivative. The thiourea group provided a UV absorbing chromophore, and the derivatives showed expectable sodium and hydrogen adducts in ion trap mass spectrometry and were suitable for liquid chromatography analysis. Reactive dithiocarbamate adducts constitute the major type of reactive ITC adduct expected in biological matrices. Incubation of a model dithiocarbamate with ammonia likewise resulted in conversion to the corresponding thiourea derivative, suggesting that a variety of matrix-bound reactive isothiocyanate adducts can be determined using this strategy. As an example of the application of the method, recovery of moringin and benzyl isothiocyanate applied to cabbage leaf discs was studied in simulated insect feeding assays. The majority of moringin was recovered as native isothiocyanate, but a major part of benzyl isothiocyanate was converted to reactive adducts.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Miriam Dantzger, Ilka Maria Vasconcelos, Valéria Scorsato, Ricardo Aparicio, Sergio Marangoni, Maria Lígia Rodrigues Macedo Herein described is the biochemical characterisation, including in vitro and in vivo assays, for a proteinase inhibitor purified from Clitoria fairchildiana seeds (CFPI). Purification was performed by hydrophobic interaction and gel filtration chromatography. Kinetic studies of the purified inhibitor showed a competitive-type inhibitory activity against bovine trypsin and chymotrypsin, with an inhibition stoichiometry of 1:1 for both enzymes. The inhibition constants against trypsin and chymotrypsin were 3.3×10?10 and 1.5×10?10M, respectively, displaying a tight binding property. SDS–PAGE showed that CFPI has a single polypeptide chain with an apparent molecular mass of 15kDa under non-reducing conditions. However, MALDI-TOF analysis demonstrated a molecular mass of 7.973kDa, suggesting that CFPI is dimeric in solution. The N-terminal sequence of CFPI showed homology with members of the Bowman–Birk inhibitor family. CFPI remained stable to progressive heating for 30min to each temperature range of 37 up to 100°C and CD analysis exhibited no changes in spectra at 207nm after heating at 90°C and subsequent cooling. Moreover, CFPI was active over a wide pH range (2–10). In contrast, reduction with DTT resulted in a loss of inhibitory activity against trypsin and chymotrypsin. CFPI also exhibited significant inhibitory activity against larval midgut trypsin enzymes from Anagasta kuehniella (76%), Diatraea saccharalis (59%) and Heliothis virescens (49%). Its insecticidal properties were further analysed by bioassays and confirmed by negative impact on A. kuehniella development.
Publication date: Available online 24 September 2015 Source:Phytochemistry Author(s): Mohamed A. Farag, Sarah T. Sakna, Nabaweya M. El-fiky, Marawan M. Shabana, Ludger A. Wessjohann Bauhinia L. (Fabaceae) comprises ca. 300–350 plant species, many of which are traditionally used in folk medicine for their antidiabetic, antioxidant and anti-inflammatory effects. Bauhinia s.l. recently has been subdivided into 9 genera based on phylogenetic data: Bauhinia s.str., Barklya, Brenierea, Gigasiphon, Lysiphyllum, Phanera, Piliostigma, Schnella (American Phanera) and Tylosema. The aerial parts of 8 species corresponding to 5 genera were analyzed: Bauhinia forficata, Bauhinia variegata, B. variegata var. candida, Bauhinia galpinii, Schnella glabra, Piliostigma racemosa, Phanera vahlii and Lysiphyllum hookeri. Leaves and shoots were subjected to metabolite profiling via UHPLC–PDA–qTOF-MS coupled to multivariate data analyzes to identify compound compositional differences. A total of 90 metabolites were identified including polyphenols and fatty acids; flavonoid conjugates accounted for most of the metabolite variation observed. This study provides a comprehensive map of polyphenol composition in Bauhinia and phytochemical species aggregations are consistent with recent Bauhinia genus taxonomic relationship derived from phylogenetic studies. DPPH radical scavenging and ?-glucosidase inhibitory assays were also performed to assess selected aspects of the antioxidant and antidiabetic potential for the examined species with respect to metabolite profiles.
Publication date: Available online 18 September 2015 Source:Phytochemistry Author(s): Yueqiu Liu, Dan Staerk, Mia N. Nielsen, Nils Nyberg, Anna K. Jäger Inhibition of the necrotizing hyaluronidase, phospholipase A2 and protease enzymes in four snake venoms by crude water and ethanol extracts of 88 plant species used against snakebites in traditional Chinese medicine was measured. High-resolution hyaluronidase inhibition profiles were constructed for the 22 plants showing highest hyaluronidase inhibition, and the results were used to guide subsequent structural analysis towards specific hyaluronidase inhibitors. Structural analysis was performed by high-performance liquid chromatography, high-resolution mass spectrometry, solid-phase extraction and nuclear magnetic resonance spectroscopy, i.e., HPLC–HRMS–SPE–NMR. This allowed identification of four non-tannin inhibitors, i.e., lansiumamide B (6) from Clausena excavata Burm.f., myricetin 3-O-?-d-glucopyranoside (7) from Androsace umbellata (Lour.) Merr., and vitexin (8) and 4?,7-dihydroxy-5-methoxyflavone-8-C-?-d-glucopyranoside (9) from Oxalis corniculata L. Absolute configuration of 2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide (1) was determined using the Mosher method, which revealed two enantiomers, i.e., (2S,3R)-2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide and (2R,3S)-2,3-dihydroxy-N-methyl-3-phenyl-N-[(Z)-styryl]propanamide with a ratio of 7:3.
Publication date: Available online 18 September 2015 Source:Phytochemistry Author(s): Yi Zang, Grégory Genta-Jouve, Tithnara Anthony Sun, Xuwen Li, Buisson Didier, Stéphane Mann, Elisabeth Mouray, Annette K. Larsen, Alexandre E. Escargueil, Bastien Nay, Soizic Prado Chemical investigation of the fungus Talaromyces stipitatus ATCC 10500, whose genome has been sequenced, led to the isolation of four undescribed talaroenamines B–E along with the known talaroenamine A. Their structures were elucidated on the basis of spectroscopic studies including mass spectrometry, extensive 2D NMR and electronic circular dichroism (ECD). Interestingly, talaroenamine A had previously been isolated from the strain of T. stipitatus ?trop C, a strain knocked out for the gene encoding a non-heme Fe(II)-dependent dioxygenase catalyzing the oxidative ring expansion leading to the tropolone, but never from a wild-type strain. All talaroenamines were evaluated for their antiplasmodial activity and Talaroenamine D exhibited the best inhibition against the chloroquine-resistant Plasmodium falciparum (FcB1 strain) without noticeable toxicity on HeLa and preadipose cell lines. In the course of the chemical investigation of T. stipitatus, an undescribed polyester was also isolated and its absolute configuration was determined by hydrolysis and transesterification followed by gas chromatography on chiral column.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Siriluk Sintupachee, Worrawat Promden, Nattaya Ngamrojanavanich, Worapan Sitthithaworn, Wanchai De-Eknamkul While attempting to isolate the enzyme geranylgeraniol 18-hydroxylase, which is involved in plaunotol biosynthesis in Croton stellatopilosus (Cs), the cDNAs for a cytochrome P450 monooxygenase (designated as CYP76F45) and an NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from the leaf. The CYP76F45 and CsCPR I genes have open reading frames (ORFs) encoding 507- and 711-amino acid proteins with predicted relative molecular weights of 56.7 and 79.0kDa, respectively. Amino acid sequence comparison showed that both CYP76F45 (63–73%) and CsCPR I (79–83%) share relatively high sequence identities with homologous proteins in other plant species. Phylogenetic tree analysis confirmed that CYP76F45 belongs to the CYP76 family and that CsCPR I belongs to Class I of dicotyledonous CPRs, with both being closely related to Ricinus communis genes. Functional characterization of both enzymes, each expressed separately in Escherichia coli as recombinant proteins, showed that only simultaneous incubation of the membrane-bound proteins with the substrate geraniol (GOH) and the coenzyme NADPH could form 8-hydroxygeraniol. The enzyme mixture could also utilize acyclic sesquiterpene farnesol (FOH) with a comparable substrate preference ratio (GOH:FOH) of 54:46. The levels of the CYP76F45 and CsCPR I transcripts in the shoots, leaves and twigs of C. stellatopilosus were correlated with the levels of a major monoterpenoid indole alkaloid, identified tentatively as 19-E-vallesamine, that accumulated in these plant parts. These results suggested that CYP97C27 and CPR I function as the enzyme geraniol-8-hydroxylase (G8H), which is likely to be involved in the biosynthesis of the indole alkaloid in C. stellatopilosus.
Publication date: Available online 11 September 2015 Source:Phytochemistry Author(s): A. Christy Hunter, Shreyal Patel, Cinzia Dedi, Howard T. Dodd, Richard A. Bryce A series of 3?,5-cycloandrostane analogues with a range of functionality (6? and 6? alcohols and ketone) at carbon 6 were tested in the endogenous lactonization pathway in Aspergillus tamarii KITA. This metabolic route converts progesterone to testololactone in high yield through a four step enzymatic pathway. To date, no studies have looked at the effect of steroids devoid of polar functionality at carbon 3 and their subsequent metabolic fate by fungi which contain Baeyer–Villiger monooxygenases. Incubation of all of the cycloandrostane analogues resulted in lactonization of ring-D irrespective of C-6 stereochemistry or absence of C-3 functionality. Presence of 6?-hydroxy group and the C-17 ketone was required in order for these analogues to undergo hydroxylation at C-15? position. All metabolites were isolated by column chromatography and were identified by 1H, 13C NMR, DEPT analysis and other spectroscopic data.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Mike Pollard, Tina M. Martin, Yair Shachar-Hill Camelina sativa is a cultivated oilseed rich in triacylglycerols containing oleic, linoleic, ?-linolenic and eicosenoic acids. As it holds promise as a model species, its lipid synthesis was characterized in vivo and in culture. Lipid accumulates at a maximum rate of about 26?g/day/seed (11.5mglipid/day/g fresh seed weight), a rate comparable with other oilseeds. Noteworthy is a late stage surge in ?-linolenic acid accumulation. Small amounts of unusual epoxy and hydroxy fatty acids are also present in the triacylglycerols. These include 15,16-epoxy- and 15-hydroxy-octadecadienoic acids and homologous series of ?7-hydroxy-alk-?9-enoic and ?9/10-hydroxy-alkanoic acids. Mid-maturation embryos cultured in vitro have growth and lipid deposition rates and fatty acid compositions that closely match that of seeds, but extended culture periods allow these rates to rise and surpass those observed in planta. Optimized thin layer chromatography systems for characterization of labeled products from acetate or glycerol labeling are described. Glycerol label is only found in acylglycerols, largely as the intact glyceryl backbone, but acetate can label acyl groups and sterols, the latter to a much higher relative specific activity. This presumably occurs because mevalonic acid precursor is derived from the non-plastid pool of acetyl-CoA that is also the source for malonyl-CoA to drive FAE1-dependent chain elongation. Particular attention has been paid to the separation of sterols and diacylglycerols, and to hydrogenation of triacylglycerols to simplify their analysis. These improved methods will allow more accurate analyses of the fluxes of lipid metabolism in cultured plant embryos.
Publication date: September 2015 Source:Phytochemistry, Volume 117 Author(s): Ruchi Badoni Semwal, Deepak Kumar Semwal, Sandra Combrinck, Alvaro M. Viljoen Gingerols 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.
Publication date: September 2015 Source:Phytochemistry, Volume 117 Author(s): Mohammad Imtiyaj Khan, P. Giridhar Betalains 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.
Publication date: September 2015 Source:Phytochemistry, Volume 117 Author(s): Mohsen Janmohammadi, Lello Zolla, Sara Rinalducci Low 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.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Anna V. Ogorodnikova, Fakhima K. Mukhitova, Alexander N. Grechkin Green tissues of spikemoss Selaginella martensii Spring possessed the complex oxylipins patterns. Major oxylipins were the products of linoleic and ?-linolenic acids metabolism via the sequential action of 13-lipoxygenase and divinyl ether synthase (DES) or allene oxide synthase (AOS). AOS products were represented by 12-oxophytodienoic acid (12-oxo-PDA) isomers. Exceptionally, S. martensii possesses high level of 12-oxo-9(13),15-PDA, which is very uncommon in flowering plants. Separate divinyl ethers were purified after micro-preparative incubations of linoleic or ?-linolenic acids with homogenate of S. martensii aerial parts. The NMR data allowed us to identify all geometric isomers of divinyl ethers. Linoleic acid was converted to divinyl ethers etheroleic acid, (11Z)-etheroleic acid and a minority of (?5Z)-etheroleic acid. With ?-linolenate precursor, the specificity of divinyl ether biosynthesis was distinct. Etherolenic and (?5Z)-etherolenic acids were the prevailing products while (11Z)-etherolenic acid was a minor one. Divinyl ethers are detected first time in non-flowering land plant. These are the first observations of fatty acid metabolism through the lipoxygenase pathway in spikemosses (Lycopodiophyta).
Publication date: September 2015 Source:Phytochemistry, Volume 117 Author(s): Maciej Ostrowski, Anna Hetmann, Anna Jakubowska The glycosylation of auxin is one of mechanisms contributing to hormonal homeostasis. The enzyme UDPG: indole-3-ylacetyl-?-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 (Km=0.52mM, Vmax=161nmolmin?1, kcat/Km=4.36mMs?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 ?-glucosidase activity, potentiated the enzyme activity at the optimal concentration of 0.3mM. 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.
Publication date: September 2015 Source:Phytochemistry, Volume 117 Author(s): Danielle Baeyens-Volant, André Matagne, Rachida El Mahyaoui, Ruddy Wattiez, Mohamed Azarkan A 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 27kDa. Far-UV CD data revealed a secondary structure content of 22% ?-helix and 26% ?-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 10mM. However, the activity was completely suppressed by Zn2+ at a concentration of 1mM. 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.
Publication date: September 2015 Source:Phytochemistry, Volume 117 Author(s): Femke de Jong, Steven J. Hanley, Michael H. Beale, Angela Karp Willow 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.
Publication date: October 2015 Source:Phytochemistry, Volume 118 Author(s): Alexander Otto, Andrea Porzel, Jürgen Schmidt, Ludger Wessjohann, Norbert Arnold The hitherto unknown natural formation of hygrophorones, antibacterial and antifungal cyclopentenone derivatives from mushrooms, was investigated for hygrophorone B12 in Hygrophorus abieticola Krieglst. ex Gröger & Bresinsky by feeding experiments in the field using 13C labelled samples of d-glucose and sodium acetate. The incorporation of 13C isotopes was extensively studied using 1D and 2D NMR spectroscopy as well as ESI-HRMS analyses. In the experiment with [U-13C6]-glucose, six different 13C2 labelled isotopomers were observed in the 2D INADEQUATE spectrum due to incorporation of [1,2-13C2]-acetyl-CoA. This labelling pattern demonstrated that hygrophorone B12 is derived from a fatty acid-polyketide route instead of a 1,4-?-d-glucan derived anhydrofructose pathway. The experiment with [2-13C]-acetate revealed an unexpected incorporation pattern in the cyclopentenone functionality of hygrophorone B12. Four single-labelled isotopomers, in particular [1-13C]-, [2-13C]-, [3-13C]-, and [4-13C]-hygrophorone B12, were detected that showed only half enrichment in comparison to the respective labelled alkyl side chain carbons. This labelling pattern indicates the formation of a symmetrical intermediate during hygrophorone B12 biosynthesis. Based on these observations, a biogenetic route via a 4-oxo fatty acid and a chrysotrione B homologue is discussed.
Publication date: September 2015 Source:Phytochemistry, Volume 117 Author(s): Patricia Bonin, Agnès Groisillier, Alice Raimbault, Anaïs Guibert, Catherine Boyen, Thierry Tonon The 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 188.8.131.52), and a mannitol-1-phosphatase hydrolyzes M1P to mannitol (EC 184.108.40.206). 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.
Posted on 11 October 2015 | 7:51 am
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