Phytochemistry (v.124, #C)

This review of the phytochemistry of seagrasses, the only higher plants living in fully marine environments, highlights the chemical diversity of secondary metabolites from seagrasses, the bioactivities of these compounds, and their functions in an ecological context.Display OmittedSeagrasses are the only higher plants living in fully marine environments; they play a significant role in coastal ecosystems. Seagrasses inhabit the coastal shelves of all continents except Antarctica and can grow in depths of up to 90 m. Because of their eminent ecological importance, innumerous studies have been dedicated to seagrasses and their ecology. However, the phytochemistry has not been equally well investigated yet and many of the existing studies in chemical ecology are only investigating the chemistry at the level of compound classes, e.g. phenolics, and not at the level of chemically defined metabolites. In the present review, the existing literature on secondary metabolites of seagrasses, their known source seagrasses, their bioactivity, and ecological function are compiled and critically assessed. Moreover, research gaps are highlighted and avenues for future research are discussed.Currently, a total of 154 chemically defined natural products have been reported from the about 70 seagrass species known worldwide. Compounds reported include simple phenols derivatives (four compounds), phenylmethane derivatives (14 compounds), phenylethane derivatives (four compounds), phenylpropane derivatives including their esters and dimers (20 compounds), chalkones (four compounds), flavonoids including catechins (57 compounds), phenylheptanoids (four compounds), one monoterpene derivative, one sesquiterpene, diterpenoids (13 compounds), steroids (31 compounds), and one alkaloid.Most of the existing bioactivity studies of seagrass metabolites and extracts have been directed to potential cytotoxic, antimicrobial, or antimacrofouling activity. Antimicrobial studies have been performed towards panels of both human pathogens and ecologically relevant pathogens. In the antimacrofouling studies, investigations of the potential of zosteric acid from the genus Zostera are the most numerous and have yielded so far the most interesting results.Studies on the chemical ecology of seagrasses often have been focused on variation in phenolic compounds and include but are not limited to studies on variation due to abiotic factors, seasonal variation, variation in response to grazing by fish or sea urchins, or following microbial attack.
Keywords: Cymodoceaceae; Hydrocharitaceae; Posidoniaceae; Zosteraceae; Chemosystematics; Marine higher plants;

Identification and characterization of two bisabolene synthases from linear glandular trichomes of sunflower (Helianthus annuus L., Asteraceae) by Anna-Katharina Aschenbrenner; Moonhyuk Kwon; Jürgen Conrad; Dae-Kyun Ro; Otmar Spring (29-37).
Two enzymes using farnesyldiphosphate to form (Z)-γ-bisabolene were identified from the RNA pool of sunflower trichomes and characterized by heterologous expression in yeast.Display OmittedSunflower is known to produce a variety of bisabolene-type sesquiterpenes and accumulates these substances in trichomes of leaves, stems and flowering parts. A bioinformatics approach was used to identify the enzyme responsible for the initial step in the biosynthesis of these compounds from its precursor farnesyl pyrophosphate. Based on sequence similarity with a known bisabolene synthases from Arabidopsis thaliana AtTPS12, candidate genes of Helianthus were searched in EST-database and used to design specific primers. PCR experiments identified two candidates in the RNA pool of linear glandular trichomes of sunflower. Their sequences contained the typical motifs of sesquiterpene synthases and their expression in yeast functionally characterized them as bisabolene synthases. Spectroscopic analysis identified the stereochemistry of the product of both enzymes as (Z)-γ-bisabolene. The origin of the two sunflower bisabolene synthase genes from the transcripts of linear trichomes indicates that they may be involved in the synthesis of sesquiterpenes produced in these trichomes. Comparison of the amino acid sequences of the sunflower bisabolene synthases showed high similarity with sesquiterpene synthases from other Asteracean species and indicated putative evolutionary origin from a β-farnesene synthase.
Keywords: Helianthus annuus L. (Asteraceae); Sunflower; Sesquiterpene biosynthesis; Bisabolene synthase; Enzyme phylogeny; Linear glandular trichome;

Cyclofarnesoids and methylhexanoids produced from β-carotene in Phycomyces blakesleeanus by Eugenio Alcalde; Humberto R. Medina; M. Mar Herrador; Alejandro F. Barrero; Enrique Cerdá-Olmedo (38-45).
First systematic analysis of the C15 and C7 apocarotenoids of Phycomyces: natural products, biosyntheses and bioconversions.Display OmittedThe oxidative cleavage of β-carotene in the Mucorales produces three fragments of 18, 15, and 7 carbons, respective heads of three families of apocarotenoids: the methylhexanoids, the trisporoids, and the cyclofarnesoids (named after their 1,6-cyclofarnesane skeleton). The apocarotenoids are easily recognized because they are absent in white mutants unable to produce β-carotene. In cultures of Phycomyces blakesleeanus we detected thirty-two apocarotenoids by LC, UV absorbance, and MS. With additional IR and NMR we identified two methylhexanoids and the eight most abundant cyclofarnesoids. Four of them were previously-unknown natural compounds, including 4-dihydrocyclofarnesine S, the most abundant cyclofarnesoid in young cultures. We arranged the apocarotenoids of the Mucorales in a scheme that helps classifying and naming them and suggests possible metabolites and biosynthetic pathways. We propose specific biosynthetic pathways for cyclofarnesoids and methylhexanoids based on structural comparisons, the time course of appearance of individual compounds, and the bioconversion of β-apo-12-carotenol, an early precursor, to three more oxygenated cyclofarnesoids by the white mutants. Some of the reactions occur spontaneously in the increasingly acidic culture media. Mating increased the contents of methylhexanoids and cyclofarnesoids by ca. threefold in young cultures and ca. twelvefold in old ones (five days); cyclofarnesine S, the most abundant cyclofarnesoid in old cultures, increased over one hundredfold. We found no differences between the sexes and no activity as sexual pheromones, but we suggest that methylhexanoids and cyclofarnesoids could mediate species-specific physiology and behavior.
Keywords: Phycomyces blakesleeanus; Phycomycetaceae; Mucorales; Bioconversion; Cyclofarnesoids; Methylhexanoids; β-Carotene; Apocarotenoids;

Dynamic metabolic changes in seeds and seedlings of Brassica napus (oilseed rape) suppressing UGT84A9 reveal plasticity and molecular regulation of the phenylpropanoid pathway by Karina Hettwer; Christoph Böttcher; Andrej Frolov; Juliane Mittasch; Andreas Albert; Edda von Roepenack-Lahaye; Dieter Strack; Carsten Milkowski (46-57).
Suppression of UGT84A9 in seeds of Brassica napus blocks the major sink pathway of sinapic acid via 1-O-sinapoylglucose (SinGlc) and affects the accumulation pattern of hydroxycinnamic acid, hydroxybenzoic acid and flavonoid conjugates in seeds and seedlings. The UV-B stress response is not impaired.Display OmittedIn Brassica napus, suppression of the key biosynthetic enzyme UDP-glucose:sinapic acid glucosyltransferase (UGT84A9) inhibits the biosynthesis of sinapine (sinapoylcholine), the major phenolic component of seeds. Based on the accumulation kinetics of a total of 158 compounds (110 secondary and 48 primary metabolites), we investigated how suppression of the major sink pathway of sinapic acid impacts the metabolome of developing seeds and seedlings. In UGT84A9-suppressing (UGT84A9i) lines massive alterations became evident in late stages of seed development affecting the accumulation levels of 58 secondary and 7 primary metabolites. UGT84A9i seeds were characterized by decreased amounts of various hydroxycinnamic acid (HCA) esters, and increased formation of sinapic and syringic acid glycosides. This indicates glycosylation and β-oxidation as metabolic detoxification strategies to bypass intracellular accumulation of sinapic acid. In addition, a net loss of sinapic acid upon UGT84A9 suppression may point to a feedback regulation of HCA biosynthesis. Surprisingly, suppression of UGT84A9 under control of the seed-specific NAPINC promoter was maintained in cotyledons during the first two weeks of seedling development and associated with a reduced and delayed transformation of sinapine into sinapoylmalate. The lack of sinapoylmalate did not interfere with plant fitness under UV-B stress. Increased UV-B radiation triggered the accumulation of quercetin conjugates whereas the sinapoylmalate level was not affected.
Keywords: Brassica napus; Brassicaceae; Metabolite profiling; Gene silencing; Glucosyltransferase; Phenylpropanoids; Sinapic acid esters; UV-B stress;

Metabolite profiles of essential oils and molecular markers analysis to explore the biodiversity of Ferula communis: Towards conservation of the endemic giant fennel by Fatma Zohra Rahali; Myriam Lamine; Mahmoud Gargouri; Iness Bettaieb Rebey; Majdi Hammami; Ibtissem Hamrouni Sellami (58-67).
Display OmittedGiant fennel (Ferula communis L.) is well known in folk medicine for the treatment of various organ disorders. The biological importance of members of genus Ferula prompted us to investigate the leaves of the endangered Tunisian medicinal plant F. communis L. not previously investigated. An estimate of genetic diversity and differentiation between genotypes of breeding germplasm is of key importance for its improvement. Thus, four F. communis populations were RAPD fingerprinted (63 RAPD markers generated by 7 primers) and the composition of their leaf essential oils (EO) (134 EO compounds) was characterized by GC–MS. Cluster analysis based on the leaf volatiles chemical composition of F. communis accessions defined three chemotypes according to main compounds have been distinguished: α-eudesmol/β-eudesmol/γ-terpinene; α-eudesmol/α-pinene/caryophyllene oxide and chamazulene/α-humulene chemotypes. A high genetic diversity within population and high genetic differentiation among them, based on RAPDs, were revealed (H pop  = 0.320 and G ST  = 0.288) caused both by the habitat fragmentation, the low size of most populations and the low level of gene flow among them. The RAPD dendrogram showed separation of three groups. Populations dominated by individuals from the β-eudesmol/γ-terpinene; chemotype showed the lowest gene diversity (H  = 0.104), while populations with exclusively α-pinene/caryophyllene oxide chemotype showed the highest value (H  = 0.285). The UPGMA dendrogram and PCA analysis based on volatiles yielded higher separation among populations, indicated specific adaptation of populations to the local environments. Correlation analysis showed a non-significant association between the distance matrices based on the genetic markers (RAPD) and chemical compounds of essential oil (P  > 0.05) indicating no influence of genetic background on the observed chemical profiles. These results reinforce the use of both volatile compounds and RAPD markers as a starting point for in situ conservation. The analysis of chemical constitution of oil of the populations from a specific region revealed predominance of specific constituents indicating possibility of their collection/selection for specific end uses like phytomedicines. Sufficient molecular and biochemical diversity detected among natural populations of this species will form the basis for the future improvement. The correlation between matrices of RAPD and essential oils was not significant. The conservation strategies of populations should be made according to their level of genetic and chemical diversity in relation to geographic location of populations. Our results give some insights into the characterization of this as yet little investigated plant.
Keywords: Ferula communis (Apiaceae); Essential oils; Metabolite profile; Chemotyping; Genetic variation; RAPD; PCoA; Hierarchical clustering analysis;

Exploring occurrence and molecular diversity of betaine lipids across taxonomy of marine microalgae by José Pedro Cañavate; Isabel Armada; José Luis Ríos; Ismael Hachero-Cruzado (68-78).
Betaine lipids were present in nine out of ten microalgae and discriminated species at the classes level. Tetraselmis suecica is the first green algae found to have DGTA.Display OmittedBetaine lipids (BL) from ten microalgae species of the kingdoms Plantae and Chromista were identified and quantified by HPLC/ESI-TOF-MS. Diacylgyceryl-N-trimethylhomoserine (DGTS) was detected in Trebouxiophyceae and Eustigmatophyceae species, whereas Tetraselmis suecica was described as the first green algae containing diacylglyceryl-hydroxymethyl-N,N,N-trimethyl-beta-alanine (DGTA). DGTA molecular species where also characterized in Cryptophyceae species as well as in the Bacillariophyceae diatom Phaeodactylum tricornutum. The Mediophyceae diatom Chaetoceros gracilis had no DGTA, but contained diacylglyceryl-carboxyhydroxymethylcholine (DGCC). A principal coordinate (PCO) analysis of microalgae species revealed the existence of three main clusters around each BL type. The first PCO axis (43.9% of total variation) grouped Chlorophyceae, Trebouxiophyceae and Eustigmatophyceae species and positively correlated with DGTS. The second PCO axis (27.8% of total variation) segregated DGTA from DGCC containing species. Cryptophyceae, Bacillariophyceae and Chlorodendrophyceae were the more closely associated species to DGTA. Mediophyceae and Dinophyceae species contained DGCC as the only BL. Molecular diversity varied from the simplest DGCC composition in Gyrodinium dorsum to the highest spectrum of ten different molecular species detected for DGTA (Rhodomonas baltica) and DGCC (C. gracilis). The fatty acid profile of DGTS was very dissimilar to that of the whole lipid cell content. DGTS from Nannochloropsis gaditana was highly unsaturated respecting to total lipids, whereas in Picochlorum atomus DGTS unsaturation was nearly one half to that of total lipids. Dissimilarity between DGTA and total lipid fatty acid profile was minimum among all BL and DGTA fatty acid unsaturation was the maximum observed in the study. New DGCC molecular species enriched in 20:5 were described in Mediophyceae diatoms. Multivariate microalgae ordination using BL as descriptors revealed a higher chemotaxonomic potential than that based on their respective BL fatty acid profile. Nevertheless, taxonomic resolution was improved when fatty acids from the whole cell lipid pool were used.
Keywords: Microalgae; Betaine lipids; Molecular species; Diacylgyceryl-N-trimethylhomoserine; Diacylglyceryl hydroxymethyl-N,N,N-trimethyl-beta-alanine; Diacylglyceryl carboxyhydroxymethylcholine; Chemotaxonomy;

Antibacterial and antifungal screening of natural products sourced from Australian fungi and characterisation of pestalactams D–F by Karren D. Beattie; Nicola Ellwood; Rohitesh Kumar; Xinzhou Yang; Peter C. Healy; Vanida Choomuenwai; Ronald J. Quinn; Alysha G. Elliott; Johnny X. Huang; Jessica L. Chitty; James A. Fraser; Matthew A. Cooper; Rohan A. Davis (79-85).
Display OmittedEighteen natural products sourced from Australian micro- or macro-fungi were screened for antibacterial and antifungal activity. This focused library was comprised of caprolactams, polyamines, quinones, and polyketides, with additional large-scale isolation studies undertaken in order to resupply previously identified compounds. Chemical investigations of the re-fermented culture from the endophytic fungus Pestalotiopsis sp. yielded three caprolactam analogues, pestalactams D–F, along with larger quantities of the known metabolite pestalactam A, which was methylated using diazomethane to yield 4-O-methylpestalactam A. The chemical structures of the previously undescribed fungal metabolites were determined by analysis of 1D/2D NMR and MS data. The structure of 4-O-methylpestalactam A was confirmed following single crystal X-ray diffraction analysis. The antibacterial and antifungal activity of all compounds was assessed, which identified three compounds, (1S,3R)-austrocortirubin, (1S,3S)-austrocortirubin, and 1-deoxyaustrocortirubin with mild activity (100 μM) against Gram-positive isolates and one compound, 2-hydroxy-6-methyl-8-methoxy-9-oxo-9H-xanthene-1-carboxylic acid, with activity against Cryptococcus neoformans and Cryptococcus gattii at 50 μM.
Keywords: Pestalotiopsis; Amphisphaeriaceae; Fungal natural products; Pestalactams A–F; Pestalactam; Caprolactam; X-ray crystal structure; Antibacterial; Antifungal; MIC screening;

Sesquiterpenoids and tirucallane triterpenoids from the roots of Scorzonera divaricata by Yong-Jin Yang; Juan Yao; Xiao-Jie Jin; Zhuan-Ning Shi; Tian-Fei Shen; Jian-Guo Fang; Xiao-Jun Yao; Ying Zhu (86-98).
Seven sesquiterpenes and triterpenes were isolated from Scorzonear divaricata. Structures were elucidated by spectroscopic data and X-ray crystallography. Their cytotoxic and antioxidant activities were assessed.Display OmittedA sulfated guaiane sesquiterpene lactone, an unusual pyridinium alkaloid with a sulfated guaiane sesquiterpene lactone nucleus, an amino conjugate of a sulfated guaiane sesquiterpene lactone, a bisabolane sesquiterpene, three tirucallane triterpenes, and six known compounds, were isolated from roots of Scorzonera divaricata. Their structures and absolute configurations were established based on chemical and spectroscopic methods, X-ray single crystal crystallography, and also by comparison with experimental and calculated ECD spectra. One of the tirucallane triterpenes exhibited significant cytotoxic activities against four human cancer cell lines (HL60, HeLa, HepG2, and SMMC-7721) in vitro. Two of sulfated guaiane sesquiterpenoids also exhibited antioxidant activities by scavenging ABTS cation free radicals. Tirucallane-type and dammarane-type triterpenes were not previously known in the genus Scorzonera. The study suggests that sulfated guaiane-type sesquiterpenoids are a valuable marker for systematic chemical studies in the Lactuceae tribe of the Asteraceae.
Keywords: Scorzonera divaricata; Asteraceae; Sesquiterpenoid; Triterpenoids; Structure elucidation; Circular dichroism; X-ray single crystal diffraction; Cytotoxicity; ABTS radical scavenging capacity;

Elucidation of hypoglycemic action and toxicity studies of insulin-like protein from Costus igneus by Manasi R. Hardikar; Mokshada E. Varma; Anjali A. Kulkarni; Prasad P. Kulkarni; Bimba N. Joshi (99-107).
The study explains the underlying mechanism of hypoglycemic activity of orally active insulin-like protein from Costus igneus, which shows sequence similarity with the predicted protein of moss.Display OmittedWe have reported earlier, an orally active insulin-like protein (ILP) from Costus igneus having potent hypoglycemic property in STZ-induced diabetic Swiss mice. The blood glucose level was reduced significantly within two hours after feeding ILP orally in an oral glucose tolerance test. The present study elucidates the mechanism underlying the hypoglycemic action of ILP. Mechanism of action of ILP was studied in differentiated L6 myotubes. 2-NBDG uptake stimulated by ILP was studied in differentiated L6 myotubes under normoglycemic, hyperglycemic and induced insulin resistant conditions. ILP treatment significantly increased 2-NBDG uptake in differentiated L6 myotubes. The levels of insulin signaling molecules IRS-1 and GLUT-4 were assessed in ILP treated L6 myotubes by immunoblot analysis of cytoplasmic and plasma membrane fractions respectively. Immunoblot analysis revealed an increase in cytoplasmic IRS-1 with a concomitant increase in GLUT-4 translocation to the plasma membrane in a time dependent manner. Toxicity studies of ILP were performed on normal as well as diabetic Swiss albino mice. ILP did not show any toxicity in the acute and sub-chronic toxicity studies in normal as well as diabetic Swiss albino mice. Mass spectrometry was carried out to identify ILP. MALDI TOF/TOF MS analysis of ILP revealed sequence homology with the predicted protein from Physcomitrella patens. Our study reveals that ILP acts via insulin signaling pathway and can be used as oral insulin mimetic.
Keywords: Costus igneus; Costaceae; Antidiabetic plants; Insulin-like protein; Insulin signaling; Diabetes mellitus; Insulin mimetics;

Prenylated phenyl polyketides and acylphloroglucinols from Hypericum peplidifolium by Serge Alain Tanemossu Fobofou; Chelsea Rebecca Harmon; Antoine Honoré Nkuete Lonfouo; Katrin Franke; Stephen M. Wright; Ludger A. Wessjohann (108-113).
Ten polyketide derivatives including previously undescribed pyranones and furans, along with known acylphloroglucinol type-compounds were isolated from the chemically unexplored Hypericum peplidifolium.Display OmittedIn search for new or chemo-taxonomically relevant bioactive compounds from chemically unexplored Hypericum species, four previously undescribed natural products, named peplidiforones A–D were isolated and characterized from Hypericum peplidifolium A. Rich., together with six known compounds. The structures of all compounds were elucidated by extensive 1D- and 2D-NMR experiments, high resolution mass spectrometric analyses (HR-MS), and by comparison with data reported in the literature. Seven of these compounds are phenyl polyketides while three are acylphloroglucinol type compounds. Peplidiforone C, which possesses an unusual carbon skeleton consisting of a furan ring substituted by a 2,2-dimethylbut-3-enoyl moiety, is the first example of a prenylated furan derivative isolated from the genus Hypericum. The cytotoxicity, antifungal, and anti-herpes simplex virus type 1 (HSV-1) activities of extracts and compounds are described.
Keywords: Hypericum peplidifolium; Hypericaceae; Polyketide derivatives; Furans; Pyranones; Prenylated natural products;