Phytochemistry (v.90, #C)

Plant epoxide hydrolases form two clades, EH1 and EH2. NbEH1.1 and NbEH2.1 from Nicotiana benthamiana are distinguished by their gene expression and different substrate specificities.Plant epoxide hydrolases (EH) form two major clades, named EH1 and EH2. To gain a better understanding of the biochemical roles of the two classes, NbEH1.1 and NbEH2.1 were isolated from Nicotiana benthamiana and StEH from potato and heterologously expressed in Escherichia coli. The purified recombinant proteins were assayed with a variety of substrates. NbEH1.1 only accepted some aromatic epoxides, and displayed the highest enzyme activity towards phenyl glycidyl ether. In contrast, NbEH2.1 displayed a broad substrate range and similar substrate specificity as StEH. The latter enzymes showed activity towards all fatty acid epoxides examined. The activity (V max) of NbEH1.1 towards phenyl glycidyl ether was 10 times higher than that of NbEH2.1. On the contrary, NbEH2.1 converted cis-9,10-epoxystearic acid with V max of 3.83 μmol min mg−1 but NbEH1.1 could not hydrolyze cis-9,10-epoxystearic acid. Expression analysis revealed that NbEH1.1 is induced by infection with tobacco mosaic virus (TMV) and wounding, whereas NbEH2.1 is present at a relatively constant level, not influenced by treatment with TMV and wounding. NbEH1.1 transcripts were present predominantly in roots, whereas NbEH2.1 mRNAs were detected primarily in leaves and stems. Overall, these two types of tobacco EH enzymes are distinguished not only by their gene expression, but also by different substrate specificities. EH1 seems not to participate in cutin biosynthesis and it may play a role in generating signals for activation of certain defence and stress responses in tobacco. However, members of the EH2 group hydrate fatty acid epoxides and may be involved in cutin monomer production in plants.
Keywords: Epoxide hydrolase; Peroxygenase; Nicotiana benthamiana; Epoxide; Diol; Triol;

Isolation and characterization of polyphenol oxidase from Sardinian poisonous and non-poisonous chemotypes of Ferula communis (L.) by Paolo Zucca; Enrico Sanjust; Martina Loi; Francesca Sollai; Mauro Ballero; Manuela Pintus; Antonio Rescigno (16-24).
Polyphenol oxidase has been extracted from poisonous and non-poisonous chemotypes of Ferula communis. The possible role of PPO in toxicity of poisonous chemotype is discussed.Display Omitted ► Ferula communis is a plant belonging to Apiaceae, widely present also in Sardinia. ► Two chemotypes, poisonous and non-poisonous of F. communis are present in the wild. ► Polyphenol oxidase was extracted, purified and characterized from both chemotypes. ► PPO from both chemotypes showed different behavior towards substrates and inhibitors. Ferula communis (L.), a plant belonging to Apiaceae, is widely present in Sardinia, Italy. Currently, interest in F. communis focuses on the presence of two chemotypes in the wild. One chemotype is poisonous to animals, whereas the other chemotype is non-poisonous. Polyphenol oxidase (PPO) has been extracted and partially purified from the two chemotypes of F. communis. The biochemical characterization of the enzymes showed significant differences. In particular, while the two PPOs were not able to use 6- and 7-hydroxycoumarin as substrates, they showed distinct specificity for 6,7- and 7,8-dihydroxycoumarin. Significant differences in the enzyme behavior towards common PPO inhibitors were also observed. In addition, activation energy and activation energy for denaturation were determined, showing significant differences between FP-PPO and FNP-PPO, particularly for denaturation kinetics. The possible roles of the two PPOs in determining differences in composition and toxicity of the two F. communis chemotypes are also discussed.
Keywords: Ferula communis; Apiaceae; Giant fennel; Poisonous chemotype; Polyphenol oxidase; Enzyme characterization; Hydroxycoumarins;

Increased accumulation of anthocyanins in Fragaria chiloensis fruits by transient suppression of FcMYB1 gene by Ariel Salvatierra; Paula Pimentel; María Alejandra Moya-León; Raúl Herrera (25-36).
Transient transformation of strawberry fruit was analyzed focusing on accumulation of anthocyanins. Lower FcMYB1 transcript level was related with a more pigmented phenotype and changes in both transcriptional profiles of structural genes of flavonoid pathway and flavonoid compounds.Display Omitted ► FcMYB1, ortholog of FaMYB1 (a suppressor of flavonoid biosynthesis) was cloned from white strawberry Fragaria chiloensis. ► Expression of FcMYB1 was higher in white strawberry than in red strawberry during fruit development. ► RNAi silencing construct depleted FcMYB1 mRNA levels in white strawberry fruits, enhancing their pigmentation. ► Fruits with lower transcript levels of this TF showed increased contents of anthocyanin and decreased levels of flavan 3-ols.Anthocyanins and proanthocyanidins (PAs), flavonoid-derived metabolites with different physiological roles, are produced by plants in a coordinated manner during fruit development by the action of transcription factors (TFs). These regulatory proteins have either an activating or repressing effect over structural genes from the biosynthetic pathway under their control. FaMYB1, a TF belonging to the R2R3-MYB family and isolated from commercial strawberry fruit (Fragaria  ×  ananassa), was reported as a transcriptional repressor and its heterologous over-expression in tobacco flowers suppressed flavonoid-derived compound accumulation. FcMYB1, an ortholog of FaMYB1 isolated from the white Chilean strawberry (Fragaria chiloensis ssp. chiloensis f. chiloensis), showed higher transcript levels in white (F. chiloensis) than in red (F. ×  ananassa cv. Camarosa) fruits. In order to assess its contribution to the discolored phenotype in F. chiloensis, FcMYB1 was transiently down-regulated in planta using an RNAi-based approach. Quantitative real-time PCR on FcMYB1 down-regulated fruits resulted an up-regulation of anthocyanidin synthase (ANS) and a strong repression of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) transcript accumulation. In addition, these fruits showed increased concentrations of anthocyanins and undetectable levels of flavan 3-ols. Altogether, these results indicate a role for FcMYB1 in regulation of the branching-point of the anthocyanin/PA biosynthesis determining the discolored phenotype of the white Chilean strawberry fruit.
Keywords: White strawberry; Fragaria chiloensis ssp. chiloensis; Rosaceae; Fruit color; RNAi silencing; Anthocyanin; Flavan 3-ol;

Metabolome profiling of floral scent production in Petunia axillaris by Naomi Oyama-Okubo; Tomoyuki Sakai; Toshio Ando; Masayoshi Nakayama; Tomoyoshi Soga (37-42).
Metabolic pathway of floral scent compounds in Petunia axillaris. Compounds whose concentrations showed nocturnal changes are in pink boxes.Emission of floral scent benzenoid/phenylpropanoid compounds in Petunia axillaris increases significantly at night, a change that is primarily determined by the endogenous concentration of these compounds in the corolla. Among wild type P. axillaris plants, there are lines that emit different amounts of scent. To understand how the nocturnal rhythm of floral scent concentrations is controlled, the concentration profiles of metabolites in the scent biosynthetic pathway in two lines of P. axillaris, a strongly scented line and a weakly scented line, are reported. In the strongly scented line, the concentration of a series of compounds from glucose-6-phosphate (G6P) to the scent compounds changed synchronously. In the weakly scented lines, the concentrations of some metabolites including 6-phosphogluconate (6PG) and downstream metabolites of shikimic acid were remarkably lower, suggesting a reduction in metabolism of G6P to 6PG and the metabolism of shikimic acid in the weakly scented line. Nocturnal increases in the concentrations of sucrose, fructose, and glucose were not found in strongly scented line. Nocturnal increases in concentrations of S-adenosylhomocysteine (SAH) and methionine and reductions in the concentrations of S-adenosylmethionine (SAM), a methylation donor to benzenoid-skeletons, were observed only in strongly scented line. It is concluded that the biosynthetic regulation of each step from G6P to the volatile scent benzenoids is performed by, at least in part, concentrations of substrates, and the regulation also affects concentrations of SAM cycle compounds.
Keywords: Petunia axillaris; Solanaceae; Metabolome analysis; Benzenoid/phenylpropanoid; Floral scent; Nocturnal rhythm;

The Δ17-desaturation involved in the conversion of eicosatetraenoic to eicosapentaenoic acids suggests that the main biosynthetic pathway leading to n-3 LC-PUFAs in Pavlova lutheri involves n-6 fatty acid precursors. The results also reflect the activity of the final desaturation and elongation steps required for n-3 LC-PUFA formation.Display Omitted► Use of 14C-radiolabeled substrates to study microalgal LC-PUFA pathways in vivo. ► Δ17-Desaturation activity involved in n-3 LC-PUFA synthesis. ► Biosynthesis pathway leading to n-3 LC-PUFA involves fatty acids of the n-6 family.The marine flagellate Pavlova lutheri is a microalga known to be rich in long-chain polyunsaturated fatty acids (LC-PUFAs) and able to produce large amounts of n-3 fatty acids, such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). As no previous study had attempted to measure the metabolic step of fatty acid synthesis in this alga, we used radiolabeled precursors to explore the various desaturation and elongation steps involved in LC-PUFA biosynthesis pathways. The incorporation of 14C-labeled palmitic ([1-14C] 16:0) and dihomo-γ-linolenic ([1-14C] 20:3n-6) acids as ammonium salts within the cells was monitored during incubation periods lasting 3, 10 or 24 h. Total lipids and each of the fatty acids were also monitored during these incubation periods. A decrease in the availability and/or accessibility of the radiolabeled substrates was observed over the incubation time. This decrease with incubation time observed using [1-14C] 16:0 and [1-14C] 20:3n-6 as substrates was used to monitor the conversion of 14C-labeled arachidonic acid ([1-14C] 20:4n-6) into longer and more unsaturated fatty acids, such as 20:5n-3 and 22:6n-3, over shorter incubation times (1 and 3 h). A metabolic relationship between the n-6 and n-3 fatty acid series was demonstrated in P. lutheri by measuring the Δ17-desaturation activity involved in the conversion of eicosatetraenoic acid to 20:5n-3. Our findings suggest that the biosynthesis pathway leading to n-3 LC-PUFA involves fatty acids of the n-6 family, which act as precursors in the biosynthesis of 20:5n-3 and 22:6n-3. This preliminary work provides a method for studying microalgal LC-PUFA biosynthesis pathways and desaturase and elongase activities in vivo using externally-radiolabeled fatty acid precursors as substrates. The use of the [1-14C] 20:4n-6 substrate also highlighted the relationships between the n-6 and the n-3 fatty acid series (e.g. Δ17-desaturation), and the final elongation and desaturation steps required for n-3 LC-PUFA formation in P. lutheri.
Keywords: Pavlova lutheri; Pavlovophyceae; Marine microalga; In vivo 14C-labeling; n-3 LC-PUFA biosynthesis; Δ17-Desaturation;

Identification of a cytochrome b5-fusion desaturase responsible for the synthesis of triunsaturated sphingolipid long chain bases in the marine diatom Thalassiosira pseudonana by Louise V. Michaelson; Jonathan E. Markham; Simone Zäeuner; Midori Matsumoto; Ming Chen; Edgar B. Cahoon; Johnathan A. Napier (50-55).
A diverged member of the cytochrome b5-fusion desaturase family has been functionally characterised as an unusual sphingolipid desaturase, responsible for the synthesis of trienes.Display Omitted► Functional characterisation of a new sphingolipid modifying enzyme in yeast. ► Identification of the enzyme responsible for generating triunsaturated LCBs. ► Addition of a new activity to the cytochrome b5 fusion desaturase family. ► Detection of orthologues in highly divergent animal species.Triunsaturated sphingolipid long chain bases (LCBs) have previously been reported in some specialised tissues of marine invertebrates. We report the presence of similar LCBs in the marine diatom Thalassiosira pseudonana and identify the cytochrome b5-fusion desaturase responsible for the introduction of the third double bond at the Δ10 position in d18:3Δ4,8,10. This study extends the catalytic repertoire of the cytochrome b5 fusion desaturase family, also indicating the presence of orthologues in other marine invertebrates. The function of these polyunsaturated sphingolipid LCBs is currently unknown but it was previously suggested that they play an essential role in primitive animals. The identification of the desaturase responsible for their synthesis paves the way for further studies.
Keywords: Desaturase; Long chain base; Sphingolipid; Thalassiosira pseudonana;

Biotransformation of clerodane diterpenoids by Rhizopus stolonifer and antibacterial activity of resulting metabolites by M. Iqbal Choudhary; Mohammad Yasin Mohammad; Syed Ghulam Musharraf; Ismail Onajobi; Akhtar Mohammad; Itrat Anis; Muhammad Raza Shah; Atta-ur-Rahman (56-61).
Transformation of clerodane lactone (1) and clerodane methyl ester (2) by Rhizopus stolonifer produced compound 4 and the isomeric pair 7 and 8, respectively. The metabolites showed moderate antibacterial activities against Gram-negative and positive bacteria.Display Omitted► Transformation of clerodane 1 and 2, by Rhizopus stolonifer, yielded metabolites 38. ► Metabolites 4, 7 and 8 were found to be new compound. ► Metabolite 8 showed a good activity against Gram-negative and positive bacteria.Microbial transformation of clerodane lactone (1) by a plant pathogen fungus, Rhizopus stolonifer, resulted in the production of metabolites 3 and 4. While incubation of clerodane methyl ester (2) by R. stolonifer yielded metabolites 58. The structures of the transformed products were determined by the spectroscopic techniques and compounds 4, 7 and 8 were found. The antibacterial activity of clerodane diterpenoids 1 and 2 and their metabolites 38 were also studied. The metabolites 37 showed moderate activities against both Gram-positive and Gram-negative organisms. While metabolite 8 showed a moderate activity against Gram-positive organisms and a good activity against Gram-negative organisms.
Keywords: Clerodane lactone; Clerodane methyl ester; Rhizopus stolonifer; Fungal transformation; Antibacterial activity;

Functional characterization of gibberellin oxidases from cucumber, Cucumis sativus L. by Maria João Pimenta Lange; Anja Liebrandt; Linda Arnold; Sara-Miriam Chmielewska; André Felsberger; Eduard Freier; Monika Heuer; Doreen Zur; Theo Lange (62-69).
Gibberellin (GA) hormones are powerful regulators of plant development. Sixteen genes encoding four different GA-oxidase families were cloned from the important crop cucumber, and their enzymatic functions were determined. The pathway drawn from the catalytic properties of these enzymes illustrate their potential for regulating GA-hormone homeostasis during cucumber development.Display Omitted► Sixteen putative GA-oxidases were identified in cucumber. ► Phylogenetic analysis distributed them into four groups (7-, 20-, 3-, and 2-oxidases). ► Members within the 7- and 2-oxidase groups have different enzymatic function. ► Members within the other two groups are functionally similar.Cucurbits have been used widely to elucidate gibberellin (GA) biosynthesis. With the recent availability of the genome sequence for the economically important cucurbit Cucumis sativus, sequence data became available for all genes potentially involved in GA biosynthesis for this species. Sixteen cDNAs were cloned from root and shoot of 3-d to 7-d old seedlings and from mature seeds of C. sativus. Two cDNAs code for GA 7-oxidases (CsGA7ox1, and -2), five for GA 20-oxidases (CsGA20ox1, -2, -3, -4, and -5), four for GA 3-oxidases (CsGA3ox1, -2, -3, and -4), and another five for GA 2-oxidases (CsGA2ox1, -2, -3, -4, and -5). Their enzymatic activities were investigated by heterologous expression of the cDNAs in Escherichia coli and incubation of the cell lysates with 14C-labelled, D2-labelled, or unlabelled GA-substrates. The two GA 7-oxidases converted GA12-aldehyde to GA12 efficiently. CsGA7ox1 converted GA12 to GA14, to 15α-hydroxyGA12, and further to 15α-hydroxyGA14. CsGA7ox2 converted GA12 to its 12α-hydroxylated analogue GA111. All five GA 20-oxidases converted GA12 to GA9 as a major product, and to GA25 as a minor product. The four GA 3-oxidases oxidized the C19-GA GA9 to GA4 as the only product. In addition, three of them (CsGA3ox2, -3, and -4) converted the C20-GA GA12 to GA14. The GA 2-oxidases CsGA2ox1, -2, -3, and -4 oxidized the C19-GAs GA9 and GA4 to GA34 and GA51, respectively. CsGA2ox2, -3, and -4 converted GA51 and GA34 further to respective GA-catabolites. In addition to C19-GAs, CsGA2ox4 also converted the C20-GA GA12 to GA110. In contrast, CsGA2ox5 oxidized only the C20 GA12 to GA110 as the sole product. As shown for CsGA20ox1 and CsGA3ox1, similar reactions were catalysed with 13-hydroxlyated GAs as substrates. It is likely that these enzymes are also responsible for the biosynthesis of 13-hydroxylated GAs in vivo that occur at low levels in cucumber.
Keywords: Cucumis sativus; Cucurbitaceae; Seedling; Seed; GA 7-oxidase (GA7ox); GA 20-oxidase (GA20ox); GA 3-oxidase (GA3ox); GA 2-oxidase (GA2ox);

The characterisation of xyloglucanase inhibitors from Humulus lupulus by Olivier Habrylo; Anne Forster; Jean-Marc Jeltsch; Vincent Phalip (70-77).
Proposed model for the role of xyloglucanase inhibitor homologues in hop. HlXEGIP1 may act in case of abiotic stress and also during biotic stress, as well HlXEHGIP2. Inhibitions of glycosyl hydrolases family 12 are shown. Physiological development may be influenced by HlXEGIPh3.Display Omitted► Three xyloglucanase inhibitors were identified and functionally described in hop. ► The three corresponding genes were differently expressed in response to stresses. ► One of them allowed xyloglucanase inhibition towards 17 fungi enzyme cocktails. ► A model proposing a different role for the three inhibitors is proposed.Phytopathogenic fungi secrete a powerful arsenal of enzymes that are potentially active against each polysaccharide component of the plant cell wall. To defend themselves, plants synthetise a variety of molecules that inhibit the activity of cell wall-degrading enzymes. Xyloglucan-specific endoglucanase inhibitor proteins (XEGIPs) act specifically against the members of fungal glycoside hydrolase family 12 (GH12 in the CAZy database). In the present study, we describe the identification of three XEGIP homologues from hop (Humulus lupulus L.). When incubating each of the recombinant inhibitors with an enzymatic cocktail from Aspergillus aculeatus (Viscozyme®), the xyloglucan-degrading endoglucanase activity decreased to 15% and 5% for HlXEGIP1 and HlXEGIP2, respectively, whereas no inhibition of the Viscozyme® enzymes was observed for the third (also called HlXEGIP homologue 3, or HlXEGIPh3). Fungal enzymatic cocktails from 20 different species also showed xyloglucan-degrading endoglucanase activities, and most of them were inhibited by HlXEGIP1 and -2. Furthermore, a real time RT-PCR analysis revealed variations in the spatial distribution of the genes encoding the three inhibitors and differential expression during development and (a) biotic stress. The role of XEGIPs in the plant-fungus interaction is discussed, and a model suggesting a distinct role of these XEGIP homologues is proposed: HlXEGIP1 may act in cases of abiotic stress, while HlXEGIP2 reacts to biotic stress, and physiological development may be influenced by HlXEGIPh3.
Keywords: Humulus lupulus L.; Cannabaceae; Hop; Biotic stress; Abiotic stress; Xyloglucanase inhibitor; XEGIP; Glycoside hydrolase family 12;

Histolocalization and physico-chemical characterization of dihydrochalcones: Insight into the role of apple major flavonoids by Matthieu Gaucher; Thomas Dugé de Bernonville; David Lohou; Sylvain Guyot; Thomas Guillemette; Marie-Noëlle Brisset; James F. Dat (78-89).
The histolocalization of dihydrochalcones in various green tissues of Malus x domestica was investigated together with their antimicrobial and iron-chelating activities. The physiological roles of this major subgroup of flavonoids in apple tree are proposed.Display Omitted► Easy staining methods for dihydrochalcone location in apple tissues are proposed. ► Dihydrochalcones localize in upper layers of leaves and around vascular bundles. ► Phloretin shows a high antimicrobial activity against a broad spectrum of microbes. ► The o-diphenol sieboldin is a potent iron-chelator.Flavonoids, like other metabolites synthesized via the phenylpropanoid pathway, possess a wide range of biological activities including functions in plant development and its interaction with the environment. Dihydrochalcones (mainly phloridzin, sieboldin, trilobatin, phloretin) represent the major flavonoid subgroup in apple green tissues. Although this class of phenolic compounds is found in very large amounts in some tissues (≈200 mg/g of leaf DW), their physiological significance remains unclear. In the present study, we highlight their tissue-specific localization in young growing shoots suggesting a specific role in important physiological processes, most notably in response to biotic stress. Indeed, dihydrochalcones could constitute a basal defense, in particular phloretin which exhibits a strong broad-range bactericidal and fungicidal activity. Our results also indicate that sieboldin forms complexes with iron with strong affinity, reinforcing its antioxidant properties and conferring to this dihydrochalcone a potential for iron seclusion and/or storage. The importance of localization and biochemical properties of dihydrochalcones are discussed in view of the apple tree defense strategy against both biotic and abiotic stresses.
Keywords: Malus x domestica; Rosaceae; Histolocalization; Antimicrobial activity; Iron chelation; Dihydrochalcones; Phloridzin; Sieboldin; Phloretin;

Sesquiterpene lactones from Lactuca canadensis and their chemotaxonomic significance by Klaudia Michalska; Edward Szneler; Wanda Kisiel (90-94).
Roots of the plant afforded 19 sesquiterpene lactones, including two new compounds (1 and 2), and 10 compounds found for the first time in Lactuca species.Display Omitted► A total of 19 sesquiterpene lactones have been isolated. ► Zaluzanin C-type guaianolides and eudesmanolides predominate. ► Two compounds are new natural products. ► Ten known compounds have no precedent in Lactuca species.A total of 19 sesquiterpene lactones were isolated from roots of Lactuca canadensis L., of which 10 were reported for the first time from Lactuca species and two were unknown. This is also the first report on the co-occurrence of three pairs of zaluzanin C-type guaianolides, epimeric at C-3, and on the presence of six eudesmanolides, oxygenated at C-1 and C-3, in Lactuca species. The new compounds were characterized as 3-epizaluzanin C-3-O-β-glucopyranoside and 11,13-dehydrolactuside C using 1D and 2D NMR and high resolution mass spectroscopy. The sesquiterpene lactone profile of this species is dominated by zaluzanin C-type guaianolides (9 compounds) and eudesmanolides (8 compounds). The dissimilarity of this profile compared to that of other taxa of the genus is discussed.
Keywords: Lactuca canadensis; Compositae; Chemosystematics; Sesquiterpene lactones;

Cytotoxic triterpenoid saponins from husks of Aesculus californica (Spach) Nutt. by Wei Yuan; Ping Wang; Zushang Su; Victoria S. Wang; Shiyou Li (95-105).
Fifteen polyhydroxyoleanene saponins were isolated from the husks of Aesculus californica. The saponins showed cytotoxicity to human non-small cell lung tumor (A549). Their structures provide important evidence for evolution and chemotaxonomy of Aesculus, a well-known intercontinental disjunct genus in the Northern hemisphere.Display Omitted► Fifteen saponins were isolated from the husks of Aesculus californica. ► These saponins have a glucopyranosyl moiety at C-3. ► These saponins provide evolutionary and taxonomic evidence for Aesculus. ► The saponins showed cytotoxicity against human non-small cell lung tumor (A549).Fifteen polyhydroxyoleanene saponins, aesculiosides C1–C15 (115), were isolated from husks of Aesculus californica. Their structures were established by extensive spectroscopic and chemical analyses. The triterpenoid saponins from A. californica have greater structural diversity than those from any other investigated species thus far in the genus Aesculus. The chemotaxonomic characteristic of aesculiosides C1–C15 is that the unit attached to the C-3 of the aglycone is a glucopyranosyl moiety, instead of a glucuronopyranosyl group in the saponins that have been isolated from other Aesculus species. The saponins isolated from A. californica then provide important evolutionary and chemotaxonomic knowledge of the Aesculus genus, a well-known intercontinental disjunct genus in the Northern hemisphere. Aesculiosides C1–C15 (115) showed cytotoxicity to human non-small cell lung tumor (A549) with GI50 ranged from 3.76 to >25 μM.
Keywords: Aesculus californica; Hippocastanaceae; Triterpenoid saponins; Cytotoxicity; Aesculioside; Chemotaxonomy;

Spirostanol saponins and esculin from Rusci rhizoma reduce the thrombin-induced hyperpermeability of endothelial cells by M. Barbič; E.A. Willer; M. Rothenhöfer; J. Heilmann; R. Fürst; G. Jürgenliemk (106-113).
Three new spirostanol saponins, six known and the coumarin esculin were isolated from Rusci rhizoma. Esculin, five isolated saponins and the aglycone neoruscogenin were tested for their ability to reduce the thrombin induced hyperpermeability of endothelial cells in vitro. Deglucoruscin, 1-O-sulpho-ruscogenin, ruscin and esculin significantly reduce the hyperpermeability at 10 μM.Display Omitted► We isolated six known and three new saponins from Rusci rhizoma.. ► Esculin is the first coumarin isolated from Butcher’s broom. ► Endothelial hyperpermeability was reduced by most isolated substances in vitro. Rusci rhizoma extracts are traditionally used against chronic venous disorders (CVD). To determine the effect of its secondary plant metabolites on the endothelium, phenolic compounds and saponins from Butcher’s broom were isolated from a methanolic extract, and their activity on the thrombin-induced hyperpermeability of human microvascular endothelial cells (HMEC-1) was investigated in vitro.In addition to the six known spirostanol saponins deglucoruscin (5), 22-O-methyl-deglucoruscoside (6), deglucoruscoside (7), ruscin (8), ruscogenin-1-O-(α-l-rhamnopyranosyl-(1 → 2)-β-d-galactopyranoside (9) and 1-O-sulpho-ruscogenin (10), three new spirostanol derivatives were isolated and identified: 3′-O-acetyl-4′-O-sulphodeglucoruscin (1), 4′-O-(2-hydroxy-3-methylpentanoyl)-deglucoruscin (2) and 4′-O-acetyl-deglucoruscin (3). Furthermore, the coumarin esculin (4), which is also prominently present in other medicinal plants used in the treatment of CVD, was isolated for the first time from Rusci rhizoma. Five of the isolated steroid derivatives (2, 5, 8, 9 and 10) and esculin (4) were tested for their ability to reduce the thrombin-induced hyperpermeability of endothelial cells in vitro, and the results were compared to those of the aglycone neoruscogenin (11). The latter compound showed a slight but concentration-dependent reduction in hyperpermeability to 71.8% at 100 μM. The highest activities were observed for the spirostanol saponins 5 and 8 and for esculin (4) at 10 μM, and these compounds resulted in a reduction of the thrombin-induced hyperpermeability to 41.9%, 42.6% and 53.3%, respectively. For 2, 5 and 8, the highest concentration tested (100 μM) resulted in a drastic increase of the thrombin effect. The effect of esculin observed at a concentration of 10 μM was diminished at 100 μM. These in vitro data provide insight into the pharmacological mechanism by which the genuine spirostanol saponins and esculin can contribute to the efficacy of Butcher’s broom against chronic venous disorders.
Keywords: Ruscus aculeatus; Endothelium; Permeability; Esculin; Spirostanol saponins;

Triterpenoid saponins from the roots of Gypsophila trichotoma Wender. by Laurence Voutquenne-Nazabadioko; Reneta Gevrenova; Nicolas Borie; Dominique Harakat; Charlotte Sayagh; Alexander Weng; Mayank Thakur; Maya Zaharieva; Max Henry (114-127).
Eleven acylated saponins were isolated from the roots of Gypsophila trichotoma. The MeOH extract show cytotoxicity on various cells line. The aminoacyl saponin enhances the cytotoxicity of the targeted toxin in HER14 cells.Display OmittedEleven triterpenoid saponins were isolated from the roots of Gypsophila trichotoma Wender. (G. trichotoma Wender. var. trichotoma) (Caryophyllaceae), together with one known compound. The structures were established on the basis of extensive NMR analysis (1H, 13C NMR, COSY, TOCSY, ROESY, HSQC, and HMBC), completed by analysis of HR-ESI-MS and ESI-MS n . The saponins have the commonly found gypsogenin as the aglycone substituted at C-3 with trisaccharide and at C-28 with oligosaccharide through a fucose residue, as saponins isolated from Gypsophila perfoliata L. originated from China. The oligosaccharide attached to C-28 is substituted with acetyl and (or) sulfate groups.Тhe cytotoxicity of the saponin extract from G. trichotoma was evaluated against a rat alveolar macrophage-like cell line NR8383 and human leukemia cell lines U937 and BV-173. The synergistic effect of the aminoacyl saponins, previously isolated from G. trichotoma, was tested for its ability to enhance the cytotoxicity of the targeted toxin in HER14 cells.
Keywords: Gypsophila trichotoma; Gypsophila perfoliata; Caryophyllaceae; Triterpenoid saponins; Cytotoxic activity;

Protoilludane sesquiterpenes from the wood decomposing fungus Granulobasidium vellereum (Ellis & Cragin) Jülich by Christina L. Nord; Audrius Menkis; Rimvydas Vasaitis; Anders Broberg (128-134).
The protoilludane sesquiterpenes 2-hydroxycoprinolone, 8-deoxy-4a-hydroxytsugicoline, 8-deoxydihydrotsugicoline, which were previously not described, radulone A and B, and coprinolone ketodiol were obtained from the wood-decay fungus Granulobasidium vellereum. The structures were elucidated with spectroscopic techniques and tested for biological activity against a selection of potential natural antagonists to G. vellereum.Display Omitted► The secondary metabolites of the fungus Granulobasidium vellereum were studied. ► Six protoilludanes were isolated, of which three were not previously described. ► The compounds were tested against potential natural antagonists to G. vellereum.The secondary metabolites of the saprotrophic wood-decay basidiomycete fungus Granulobasidium vellereum were studied. Six sesquiterpenes were obtained; 2-hydroxycoprinolone (1), 8-deoxy-4a-hydroxytsugicoline (2), 8-deoxydihydrotsugicoline (3), which were previously not described, radulone A and B, and coprinolone ketodiol. Additionally, base-treatment of 1 yielded the diagnostic degradation products 1a and 1b, whereas radulone A was found to form 4 under mild acidic conditions. The structures were determined by NMR, MS, CD and polarimetry, along with biosynthetic considerations. Radulone A fully inhibited the spore germination of the potentially competing fungi Phlebiopsis gigantea, Coniophora puteana and Heterobasidion occidentale at 10 μM, 500 μM and 100 μM, respectively. None of the other substances tested gave rise to any growth inhibition.
Keywords: Cyphellaceae; Granulobasidium vellereum; Structural elucidation; Sesquiterpenes; Protoilludane;

Vitamin A content recorded in different parts of Cymodocea serrulata in different seasons. Maximum amount of Vitamin A was registered in the rhizome of Cymodocea during the postmonsoon of 2010–11 and 2011–12, respectively.Display Omitted► First report on certain seagrass phytochemical. ► Dietary fiber highest in rhizome of Halophila. ► Highest K (Cymodocea), Ca & Mg (Halodule) and Na (Syringodium). ► Max vit A (rhizome), C & E (root) of Cymodocea and B3 (Syringodium, root). ► Max amount of amino acids in Halodule and of fatty acids in Syringodium.The amount of dietary fibre, mineral and vitamin were determined in root, rhizome and leaf of four commonly-available seagrasses, Cymodocea serrulata, Syringodium isoetifolium, Halophila ovalis and Halodule pinifolia at a station off Hare Island, Tuticorin (8°45′ N, 78°12′ E) in the Gulf of Mannar Biosphere region during premonsoon (July–September), monsoon (October–December) and postmonsoon (January–March) seasons of 2010–2011 and 2011–2012 study period. The entire tissues from each seagrass were subjected to HPLC and GC analysis for determining amino acid and fatty acid profiles respectively. The rhizomes of H. ovalis possessed highest amount of dietary fibre during monsoon. C. serrulata showed maximum content of K in rhizome during monsoon. Highest amount of Ca and Mg was recorded in the rhizome and leaf of H. pinifolia in postmonsoon. S. isoetifolium exhibited peak value for Na in its rhizome during monsoon. Highest amounts of Vitamin A, C and E were registered in the rhizome/root of Cymodocea during postmonsoon. Vitamin B3 was maximum in the root of Syringodium in monsoon. Eighteen of the twenty amino acids detected in seagrasses were found to the maximum level in Halodule. Syriingodium showed the highest amount of six of the seven fatty acids recorded.
Keywords: Seagrasses; Dietary fibres; Minerals; Vitamins; Amino acids; Fatty acids;

Cytotoxic constitutents from Cryptocarya maclurei by Rui Feng; Ting Wang; Wei Wei; Ren Xiang Tan; Hui Ming Ge (147-153).
Flavonoids, cryptogiones G–K, and a polyketide, cryptomaclurone, were isolated from the stems of Cryptocarya maclurei. A putative biosynthetic pathway to these compounds was proposed. Cryptomaclurone exhibited moderate cytotoxicity.Display Omitted► Phytochemical constituents of Cryptocarya maclurei were investigated. ► Five flavonoids and a polyketide were isolated. ► A putative biosynthetic pathway to the isolates was proposed. ► The polyketide exhibited moderate cytotoxicity.A phytochemical study of Cryptocarya maclurei led to isolation of five flavanones, cryptogiones G–H, and a polyketide, cryptomaclurone. The structures of the isolates were elucidated by analysis of the 1D and 2D NMR spectroscopic data, and their absolute configurations were determined by CD methods. A putative biosynthetic pathway to them is proposed. Cytotoxicity of these compounds evaluated against KB, SGC-7901 and SW 1116 cancer cell lines, with only cryptomaclurone exhibiting moderate cytotoxicity (IC50 28.2, 28.4 and 16.4 μM, respectively).
Keywords: Cryptocarya maclurei; Lauraceae; Flavonoid; Polyketide; Biosynthetic pathway; Cytotoxicity;