Phytochemistry (v.82, #C)

Four homologues of alanine aminotransferase have been isolated from wheat seedlings. Two of them, GGAT1 and GGAT2, participate in photorespiration, whereas the other two appear to be involved mainly in alanine metabolism. All of them are active as monomers.Display Omitted► Highly purified preparations of four wheat AlaAT homologues were successfully achieved. ► In native state AlaAT homologues are active as monomers. ► Kinetic analysis of GGATs suggests their involvement in photorespiratory metabolism.Four homologues of alanine aminotransferase have been isolated from shoots of wheat seedlings and purified by saline precipitation, gel filtration, preparative electrophoresis and anion exchange chromatography on Protein-Pak Q 8HR column attached to HPLC. Alanine aminotransferase 1 (AlaAT1) and 2 (AlaAT2) were purified 303- and 452-fold, respectively, whereas l-glutamate: glyoxylate aminotransferase 1 (GGAT1) and 2 (GGAT2) were purified 485- and 440-fold, respectively. Consistent inhibition of AlaAT (EC 2.6.1.2) and GGAT (EC 2.6.1.4) activities by p-hydroxymercuribenzoate points on participation of cysteine residues in the enzyme activity. The molecular weight of AlaAT1 and AlaAT2 was estimated to be 65 kDa and both of them are monomers in native state. Nonsignificant differences between K m using alanine as substrate and catalytic efficiency (k cat/K m) for l-alanine in reaction with 2-oxoglutarate indicate comparable kinetic constants for AlaAT1 and AlaAT2. Similar kinetic constants for l-alanine in reaction with 2-oxoglutarate and for l-glutamate in reaction with pyruvate for all four homologues suggest equally efficient reaction in both forward and reverse directions. GGAT1 and GGAT2 were able to catalyze transamination between l-glutamate and glyoxylate, l-alanine and glyoxylate and reverse reactions between glycine and 2-oxoglutarate or pyruvate. Both GGATs also consisted of a single subunit with molecular weight of about 50 kDa. The estimated K m for GGAT1 (3.22 M) and GGAT2 (1.27 M) using l-glutamate as substrate was lower in transamination with glyoxylate than with pyruvate (9.52 and 9.09 mM, respectively). Moreover, distinctively higher values of catalytic efficiency for l-glutamate in reaction with glyoxylate than for l-glutamate in reaction with pyruvate confirm involvement of these homologues into photorespiratory metabolism.
Keywords: Alanine aminotransferase; Glutamate: glyoxylate aminotransferase; Homologues; Wheat;

A dehydrogenative polymer (DHP) was synthesized in vitro through dehydrogenative polymerization using different ratios of sinapyl alcohol (SA) and coniferyl alcohol (CA) (10:0, 8:2, 6:4, 2:8, 0:10), respectively..Display Omitted► Oxidizability of coniferyl alcohol (CA) and sinapyl alcohol (SA) for the peroxidases were estimated in the order of Coprinus cinereus peroxidase (CiP) > soybean peroxidase (SBP) > horseradish peroidase (HRP), SBP > CiP > HRP, respectively. ► The dehydrogenative polymerisates (DHP) yields were DHP-C (9–72%) ≈ DHP-H (7–69%) > DHP-S (0–27%). ► The average molecular weights of the DHPs were between 3,400 and 5,000 Da, which was only ca. 1/2–1/3 that of native MWL. ► The frequency of the β-O-4 linkage formation, especially with CA, was influenced by type of peroxidases used.In this study, dehydrogenative polymers (DHP) were synthesized in vitro through dehydrogenative polymerization using different ratios of coniferyl alcohol (CA) and sinapyl alcohol (SA) (10:0, 8:2, 6:4, 2:8, 0:10), in order to investigate the monolignol coupling mechanism in the presence of horseradish peroxidase (HRP), Coprinus cinereus peroxidase (CiP) or soybean peroxidase (SBP) with H2O2, respectively. The turnover capacities of HRP, CiP and SBP were also measured for coniferyl alcohol (CA) and sinapyl alcohol (SA), and CiP and SBP were found to have the highest turnover capacity for CA and SA, respectively. The yields of HRP-catalyzed DHP (DHP-H) and CiP-catalyzed DHP (DHP-C) were estimated between ca. 7% and 72% based on the original weights of CA/SA in these synthetic conditions. However, a much lower yield of SBP-catalyzed DHP (DHP-S) was produced compared to that of DHP-H and DHP-C. In general, the DHP yields gradually increased as the ratio of CA/SA increased. The average molecular weight of DHP-H also increased with increasing CA/SA ratios, while those of DHP-C and DHP-S were not influenced by the ratios of monolignols. The frequency of β-O-4 linkages in the DHPs decreased with increasing CA/SA ratios, indicating that the formation of β-O-4 linkages during DHP synthesis was influenced by peroxidase type.
Keywords: Dehydrogenative polymer; Peroxidases; Coniferyl alcohol; Sinapyl alcohol; Turnover capacity; DFRC analysis;

Arogenate dehydratase (ADT) was differentially knocked out in a series of mutant lines. The impact on free amino acid pools was determined with the most significant effect being on Phe pool sizes in lignifying stems.Display Omitted► Twenty-five Arabidopsis thaliana arogenate dehydratase mutants were analyzed for amino acid pool sizes. ► Those mutants, which had ADT directed to Phe conversion into stem lignin, had much reduced Phe levels. ► No other significant effects on amino acid pool sizes were observed.Phe is formed from arogenate in planta through the action of arogenate dehydratase (ADT), and there are six ADT isoenzymes in the “model” vascular plant species Arabidopsis thaliana. This raised the possibility that specific ADTs may be differentially regulated so as to control Phe biosynthesis for protein synthesis vs its much more massive deployment for phenylpropanoid metabolism. In our previous reverse genetics study using 25 single/multiple ADT knockout (KO) lines, a subset of these knockouts was differentially reduced in their lignin contents.In the current investigation, it was hypothesized that Phe pool sizes might correlate well with reduction in lignin contents in the affected KO lines. The free amino acid contents of these KO lines were thus comprehensively analyzed in stem, leaf and root tissues, over a growth/developmental time course from 3 to 8 weeks until senescence. The data obtained were then compared to, and contrasted with, the differential extent of lignin deposition occurring in the various lines. Relative changes in pool sizes were also analyzed by performing a pairwise confirmatory factor analysis for Phe:Tyr, Phe:Trp and Tyr:Trp, following determination of the deviation from the mean for Phe, Tyr and Trp in each plant line. It was found that the Phe pool sizes measured were differentially reduced only in lignin-deficient lines, and in tissues and at time points where lignin biosynthesis was constitutively highly active (in wild type lines) under the growth conditions employed. In contrast, this trend was not evident across all ADT KO lines, possibly due to maintenance of Phe pools by non-targeted isoenzymes, or by feedback mechanisms known to be in place.
Keywords: Arabidopsis thaliana; Brassicaceae; Arogenate dehydratase; Knockout mutants; Phenylalanine; Tyrosine; Tryptophan; Amino acids; Lignins; Phenylpropanoids;

RIKEN tandem mass spectral database (ReSpect) for phytochemicals: A plant-specific MS/MS-based data resource and database by Yuji Sawada; Ryo Nakabayashi; Yutaka Yamada; Makoto Suzuki; Muneo Sato; Akane Sakata; Kenji Akiyama; Tetsuya Sakurai; Fumio Matsuda; Toshio Aoki; Masami Yokota Hirai; Kazuki Saito (38-45).
The ReSpect database is composed of tandem mass spectrometry (MS/MS) data, and the web applications are established for the annotation of untargeted MS/MS data.Display Omitted► RIKEN tandem mass spectral database (ReSpect) is MS/MS data resource and database for phytochemicals. ► The ReSpect database is primarily composed of MS/MS data obtained from the available literature. ► An MS/MS fragment search system was established for the annotation of untargeted MS/MS data. ► All web application and data resource in ReSpect is publicly available.The fragment pattern analysis of tandem mass spectrometry (MS/MS) has long been used for the structural characterization of metabolites. The construction of a plant-specific MS/MS data resource and database will enable complex phytochemical structures to be narrowed down to candidate structures. Therefore, a web-based database of MS/MS data pertaining to phytochemicals was developed and named ReSpect (RIKEN tandem mass spectral database). Of the 3595 metabolites in ReSpect, 76% were derived from 163 literature reports, whereas the rest was obtained from authentic standards. As a main web application of ReSpect, a fragment search was established based on only the m/z values of query data and records. The confidence levels of the annotations were managed using the MS/MS fragmentation association rule, which is an algorithm for discovering common fragmentations in MS/MS data. Using this data resource and database, a case study was conducted for the annotation of untargeted MS/MS data that were selected after quantitative trait locus analysis of the accessions (Gifu and Miyakojima) of a model legume Lotus japonicus. In the case study, unknown metabolites were successfully narrowed down to putative structures in the website.
Keywords: MS/MS; Metabolomics; Annotation; Database; Similarity search; MS/MS fragmentation association rule;

Polyethylene glycol treatment promotes metabolic events associated with maize callus morphogenic competence by Anatoliy V. Lygin; Manal M. Abdel-Rahman; Alexander V. Ulanov; Jack M. Widholm; Vera V. Lozovaya (46-55).
PEG application over long culture period changed metabolism of nonregenerable maize calli so they acquire biochemical characteristics of regenerable calli and capacity to regenerate plants.Display Omitted► Amendment of PEG into culture medium has transformed non-regenerable maize callus to regenerable callus. ► Both primary and secondary callus metabolism was modified by PEG. ► Metabolic markers of regenerability in vitro were identified.Metabolic changes were studied, which accompanied the conversion of 6 month old HiII maize non-regenerable (NR) calli into regenerable (R) calli when cultured for 63 days with 10% polyethylene glycol (PEG) (3350 MW) in culture medium.The conversion of 6 month old NR to R callus morphotype caused by PEG application decreased cell wall contents in callus dry mass and changed cell wall phenolics making their profile similar to that of R callus by reduction of lignin and ester- and ether-bound phenolic concentrations, including p-coumaric acid and ester- and ether-bound diferulates and by increase of the ratios of ester- and ether-bound ferulic acid/coumaric acid and ferulic acid/diferulic acid in cell walls of NR callus. Some similar changes of cell wall phenolics caused by PEG application were also found in 48 month old NR callus, that changed the morphology, but did not regenerate plants. However, there were no changes in the old callus in levels of total ester and ether-bound cell wall phenolics and substantially smaller decreases were found in ratios of ester- and ether-bound ferulic acid/coumaric acid and ferulic acid/diferulic acid, as well as in diferulate concentrations compared to young NR callus cultured with PEG. Remarkably, application of PEG also changed the primary metabolism of young NR callus tissues, so that they acquired metabolic features of highly regenerable callus. These data clearly suggest that PEG alters metabolism of NR calli, so they acquire biochemical characteristics of R calli, and that adaptive osmotic adjustments vary in different types of callus tissues.
Keywords: Maize (Zea mays L.); Gramineae; Tissue culture; Cell wall phenolics; Metabolome; Polyethylene glycol; Morphogenic potential;

Microbial transformation of ursolic acid by Syncephalastrum racemosum (Cohn) Schroter AS 3.264 by Fei-xia Huang; Wen-zhi Yang; Fei Ye; Jin-ying Tian; Hong-bo Hu; Lin-min Feng; De-an Guo; Min Ye (56-60).
Five triterpenoids were obtained from the biotransformation of ursolic acid by Syncephalastrum racemosum AS 3.264. One showed moderate PTP1B inhibitory activity.Display Omitted► Biotransformation of ursolic acid by S. racemosum yielded five triterpenoids. ► The catalytic reactions included hydroxylation, carbonylation, and condensation. ► The condensation to form a lactone or epoxy ring is unusual for biotransformation. ► One compound showed moderate PTP1B inhibitory activity.Biotransformation of ursolic acid by the filamentous fungus Syncephalastrum racemosum (Cohn) Schroter AS 3.264 yielded five metabolites. Their structures were identified as 3β,21β-dihydroxy-urs-11-en-28-oic acid-13-lactone, 3β,7β,21β-trihydroxy-urs-11-en-28-oic acid-13-lactone, 1β,3β-dihydroxy-urs-12-en-21-one-28-oic acid, 1β,3β,21β-trihydroxy-urs-12-en-28-oic acid and 11,26-epoxy-3β,21β-dihydroxy-urs-12-en-28-oic acid based on NMR and MS spectroscopic analyses. The condensation reactions to form 28-oic acid-13-lactone ring and 11,26-epoxy ring are not frequently seen for the biotransformation of triterpenoids. One compound showed moderate inhibitory activity against protein tyrosine phosphatase 1B (PTP1B).
Keywords: Biotransformation; PTP1B; Syncephalastrum racemosum; Triterpenoids; Ursolic acid;

The potential of Cyathus africanus for transformation of terpene substrates by Kayanne P. McCook; Avril R.M. Chen; William F. Reynolds; Paul B. Reese (61-66).
Incubation of sesqui-, di- and triterpenes with the fungus, in the presence of chlorocholine chloride, led to the generation of products of hydroxylation, epoxidation, hydrolysis as well as Wagner–Meerwein rearrangement.Display Omitted► Investigation of ability of Cyathus sp. to functionalise terpenes. ► Use of pulse feeding protocol to induce cytochrome P450 enzymes. ► First report in which terpenes hydroxylated successfully by Cyathus africanus. ► C. africanus executed Wagner–Meerwein rearrangement on triterpene.The insecticidal sesquiterpenes cadina-4,10(15)-dien-3-one and aromadendr-1(10)-en-9-one were administered to the fungus Cyathus africanus ATCC 35853. Biotransformation of the former produced (4R)-9α-hydroxycadin-10(15)-en-3-one, while the latter gave 2β-hydroxyaromadendr-1(10)-en-9-one, 2α-hydroxyaromadendr-1(10)-en-9-one and 10α-hydroxy-1β,2β-epoxyaromadendran-9-one. The bioconversion of santonin led to the production of two analogues, 11,13-dihydroxysantonin and the hitherto unreported 8α,13-dihydroxysantonin, while cedrol yielded 3β,8β-dihydroxycedrane and 3α,8β-dihydroxycedrane. Stemod-12-ene, a diterpene, was transformed to 2-oxostemar-13-ene, a hitherto unknown analogue with a rearranged carbon framework. When methyl betulonate, a triterpenoid belonging to the lupane family, was supplied to the fungus 18α-ursane and 18α-oleanane derivatives, namely 19β-hydroxy-3-oxo-18α-oleanan-28-oic acid and 19α-hydroxy-3-oxo-18α-ursan-28-oic acids, were generated. There are no previous reports of fungal transformation of a triterpene in which a skeletal rearrangement occurred. All substrate administration experiments were done in the presence of the terpene cyclase inhibitor chlorocholine chloride (CCC), using the single phase – pulse feed method.
Keywords: Cyathus africanus; Biotransformation; Hydroxylation; Rearrangement; Chlorocholine chloride;

Polyphenol metabolism provides a screening tool for beneficial effects of Onobrychis viciifolia (sainfoin) by Jana Thill; Ionela Regos; Mohamed A. Farag; Asma F. Ahmad; Justyna Kusek; Ana Castro; Karin Schlangen; Christine Hayot Carbonero; Ilya Z. Gadjev; Lydia M.J. Smith; Heidi Halbwirth; Dieter Treutter; Karl Stich (67-80).
The spectrum of soluble compounds is largely determined by the different substrate specificities of dihydroflavonol 4-reductase and flavonol synthase. The ratio of these enzyme activities is a promising screening tool for future sainfoin breeding.Display Omitted► Dihydroflavonol 4-reductase (DFR) does not accept dihydrokaempferol as substrate. ► Divergent substrate acceptance of flavonol synthase and DFR promotes flavonol formation. ► DFR:FLS activity ratio provides a screening tool for germplasm collection. ► Flavonols and flavan 3-ols were most suitable for the discrimination of accessions. ► The geographical origin is not reflected in the contents of soluble polyphenols. Onobrychis viciifolia (sainfoin) is a traditional fodder legume showing multiple benefits for the environment, animal health and productivity but weaker agronomic performance in comparison to other legumes. Benefits can be mainly ascribed to the presence of polyphenols. The polyphenol metabolism in O. viciifolia was studied at the level of gene expression, enzyme activity, polyphenol accumulation and antioxidant activity. A screening of 37 accessions regarding each of these characters showed a huge variability between individual samples. Principal component analysis revealed that flavonols and flavan 3-ols are the most relevant variables for discrimination of the accessions. The determination of the activities of dihydroflavonol 4-reductase and flavonol synthase provides a suitable screening tool for the estimation of the ratio of flavonols to flavan 3-ols and can be used for the selection of samples from those varieties that have a specific optimal ratio of these compounds for further breeding.
Keywords: Onobrychis viciifolia (sainfoin); Soluble polyphenols; Flavonoids; Flavonols; Flavan 3-ols; Dihydroflavonol 4-reductase substrate specificity; Flavonoid enzymes; Flavonoid genes; Screening tool;

Differences in volatile terpene composition between the bark and leaves of tropical tree species by Elodie A. Courtois; Christopher Baraloto; C.E. Timothy Paine; Pascal Petronelli; Pierre-Alain Blandinieres; Didier Stien; Emeline Höuel; Jean-Marie Bessière; Jérôme Chave (81-88).
A study on 55 tropical tree species indicate that bark hold a distinct and more diverse blend of volatile terpenes than leaves.Display Omitted► We measured volatile terpenes in bark and leaves of 55 tropical tree species. ► The blend released by bark is more diverse than that released by leaves. ► Bark terpene composition is different than leaves terpene composition.Volatile terpenes are among the most diverse class of defensive compounds in plants, and they are implicated in both direct and indirect defense against herbivores. In terpenes, both the quantity and the diversity of compounds appear to increase the efficiency of defense as a diverse blend of compounds provides a more efficient protection against a broader range of herbivores and limits the chances that an enemy evolves resistance. Theory predicts that plant defensive compounds should be allocated differentially among tissues according to the value of the tissue, its cost of construction and the herbivore pressure on it. We collected volatile terpenes from bark and leaves of 178 individual tree belonging to 55 angiosperm species in French Guiana and compare the kind, amount, and diversity of compounds in these tissues. We hypothesized that in woody plants, the outermost part of the trunk should hold a more diverse blend of volatile terpenes. Additionally, as herbivore communities associated with the leaves is different to the one associated with the bark, we also hypothesized that terpene blends should be distinct in the bark vs. the leaves of a given species. We found that the mixture of volatile terpenes released by bark is different and more diverse than that released by leaves, both in monoterpenes and sesquiterpenes. This supports our hypothesis and further suggests that the emission of terpenes by the bark should be more important for trunk defense than previously thought.
Keywords: French Guiana; Herbivory; Optimal defense theory; Secondary metabolites; Wood;

Lignans in seeds of Linum species by Thomas J. Schmidt; Michael Klaes; Jandirk Sendker (89-99).
The lignan profiles of seeds of 23 Linum taxa as determined by HPLC/ESI-MSMS are described and compared. Two major groups of species exist that accumulate cyclolignans mainly of the aryltetralin- and arylnaphthalene/dihydronaphthalene types, respectively. The latter also contain 8R,8′R-secoisolariciresinol diglucoside whereas the former were neither found to accumulate this lignan nor its 8S,8′S-stereoisomer. The occurrence of this latter lignan is restricted to L. usitatissimum and some of its close relatives, which, on the other hand, do not accumulate significant amounts of non-polar cyclolignans.This distribution of lignans correlates strongly with the molecular phylogeny of the genus Linum. Chemosystematic and biosynthetic aspects are discussed.Display Omitted► Seeds of 23 Linum taxa were analyzed for non-polar as well as polar lignans by HPLC/ESI-MSMS. ► The overall distribution of 23 lignans (some aryltetralin esters) correlates with phylogeny. ► 8R,8′R-SDG co-occurs with arylnaphthalene-/dihydronaphthalene cyclolignans. ► 8S,8′S-SDG is restricted to the L. usitatissimum group (insignificant amounts of cyclolignans). ► Seeds of taxa accumulating aryltetralin lignans do not contain SDG in either form.Mature seeds of 20 Linum species were analyzed for their content of lignans. The seeds of common flax (Linum usitatissimum L.) are known to contain as characteristic lignan sesoisolariciresinol diglucoside (SDG), whose presence in seeds of some other Linum species has also been reported. In order to investigate the material for the presence of such very polar lignans as well as for less polar non-glycosidic lignans as frequently found in aerial parts of Linum species, polar and non-polar extracts of each sample were analyzed by HPLC/ESI-MSMS.SDG was detected in 15 of 16 investigated seed samples of taxa representing sections Linum and Dasylinum. None of eight samples of taxa from sections Syllinum and Linopsis contained detectable amounts of SDG. Quite interestingly, most of the SDG-positive samples contained the 8R,8′R-isomer exclusively while only three (including L. usitatissimum) contained the 8S,8′S-stereoisomer as the predominant form. As a most noteworthy finding, the dichloromethane extracts obtained from seeds of several Linum species were found to contain significant concentrations of non-polar cyclolignans of the arylnaphthalene/-dihydronaphthalene lactone type or, alternatively of the aryltetralin lactone type. Thus, seeds of Linum perenne L. as well as those of several other representatives of sections Linum and Dasylinum were found to contain significant concentrations of the arylnaphthalene justicidin B along with further compounds of this type and some aryldihydronaphthalene-type lignans. On the other hand, seeds of Linum flavum and further representatives of section Syllinum were found to contain aryltetralin-type lignans, mainly in the form of esters with aliphatic carboxylic acids, such as 6-methoxypodophyllotoxin-7-O-n-hexanoate, whose occurrence in L. flavum seeds has very recently been reported by us for the first time.Various chemosystematic and biogenetic aspects are discussed in the light of these results.
Keywords: Linum; Linaceae; Flaxseed; Lignan; Arylnaphthalene; Aryltetralin; Secoisolariciresinol diglucoside (SDG); HPLC/ESI-MSMS;

Anti-hepatitis B virus constituents from the stem bark of Streblus asper by Jun Li; Yan Huang; Xin-Lan Guan; Jian Li; Sheng-Ping Deng; Qiang Wu; Yan-Jun Zhang; Xiao-Jian Su; Rui-Yun Yang (100-109).
The structures and absolute stereochemistries of seven neolignans including compounds (14) isolated from stem bark of Streblus asper have been determined by detailed spectroscopic analyses. The anti-HBV activity assays of the isolated compounds indicated that magnolol and 9-β-xylopyranosyl-isolariciresinol were the most active against the hepatitis B virus.Display Omitted► The constituents of stem bark of Streblus asper were investigated. ► Activity-guided isolation led to discovery of 22 compounds including 7 previously unreported. ► 10 compounds exhibited anti-HBV activities in vitro. ► Magnolol and 9-β-xylopyranosyl-isolariciresinol showed significant anti-HBV activities.Seven compounds, (7′S,8′S)-trans-streblusol A, (7′R,8′S)-erythro-streblusol B, (7′S,8′S)-threo-streblusol B, 8′R-streblusol C, streblusquinone, (8R,8′R)-streblusol D, and streblusol E, along with 15 known compounds (822) were isolated from the n-butanol-soluble part of the MeOH extract of stem bark of Streblus asper. Their structures were elucidated through application of extensive spectroscopic methods, including ESI-MS and 2D NMR spectroscopy (HMQC and HMBC). The stereochemistry at the chiral centers was determined using CD spectra, as well as analyses of coupling constants and optical rotation data. The isolated lignans and allylbenzene derivatives were evaluated for their anti-HBV activities in vitro using the HBV transfected Hep G2.2.15 cell line. The most active compounds, magnolol and 9-β-xylopyranosyl-isolariciresinol, exhibited significant anti-HBV activities with IC50 values of 2.03 and 6.58 μM for secretion of HBsAg, with no cytotoxicity, and of 3.76 and 24.86 μM for secretion of HBeAg, with no cytotoxicity.
Keywords: Streblus asper; Moraceae; (7′S,8′S)-trans-streblusol A; Streblusquinone; Streblusol E; Anti-HBV;

Bioactive constituents of Clausena lansium and a method for discrimination of aldose enantiomers by De-Yang Shen; Chih-Hua Chao; Hsiu-Hui Chan; Guan-Jhong Huang; Tsong-Long Hwang; Chin-Yu Lai; Kuo-Hsiung Lee; Tran Dinh Thang; Tian-Shung Wu (110-117).
Glycosides, clausenosides A and B, and carbazole alkaloids, clausenaline A, claulamine A, and claulamine B, were isolated from the stems of Clausena lansium. Claulamine A has an 1-oxygenated carbazole framework with a rare 2,3-lactone ring. Claulamine B represents an unusual acetal carbazole alkaloid.Display Omitted► Alkyl glycosides and carbazole alkaloids were isolated from Clausena lansium. ► Thirty-one compounds were evaluated for anti-inflammatory activity. ► A biosynthetic pathway to carbazole alkaloids in C. lansium is proposed. ► A practical simultaneous analysis of aldose enantiomers was also developed.Glycosides, clausenosides A and B, and carbazole alkaloids, clausenaline A, claulamine A, and claulamine B, together with 50 known compounds, were isolated from the stems of Clausena lansium. Their structures were determined by means of spectroscopic methods, including that of CD and 1D/2D NMR analysis. Claulamine A has a 1-oxygenated carbazole skeleton with a rare 2,3-lactone ring, and claulamine B represents an hitherto unknown acetal carbazole alkaloid. Thirty-one of the isolated known compounds were evaluated in various assays for anti-inflammatory activity. Among them, imperatorin, isoheraclenin, and osthol exhibited selective and potent inhibition of formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation, and lansiumarin C also decreased nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production in lipopolysaccharide (LPS)-induced macrophages. In addition, a modified HPLC method of pre-column derivatization was developed that is more practical for simultaneous analysis of aldose enantiomers as compared to the literature method. The absolute configurations of the sugar moieties in clausenosides A and B were determined with this modified method.
Keywords: Clausena lansium; Rutaceae; Aldose enantiomers; Anti-inflammatory; Cytotoxicity;

Secondary metabolites from the stems of Engelhardia roxburghiana and their antitubercular activities by Ho-Chen Wu; Ming-Jen Cheng; Chien-Fang Peng; Shyh-Chyun Yang; Hsun-Shuo Chang; Chu-Hung Lin; Chyi-Jia Wang; Ih-Sheng Chen (118-127).
Four diarylheptanoids, two cyclic diarylheptanoids, one naphthoquinone dimer, one 1-tetralone, and 24 known compounds were isolated from stems of Engelhardia roxburghiana. The structures of 18 were identified by means of spectroscopic analysis.Display Omitted► Eight new compounds were isolated from stems of Engelhardia roxburghiana. ► The structures of new compounds 18 were determined by spectroscopic analysis. ► Compounds 5, 6, 13, 17, 22, and 23 showed antitubercular activity.Bioassay-guided fractionation of stems of Engelhardia roxburghiana led to isolation of: four diarylheptanoids, engelheptanoxides A–D (14); two cyclic diarylheptanoids, engelhardiols A (5) and B (6); one naphthoquinone dimer, engelharquinonol (7); and one 1-tetralone, (4S)-4,6-dihydroxy-1-tetralone (8), along with 24 known compounds (932). The structures of 18 were by spectroscopic analysis. Compounds 5, 6, 13, 22, and 23 showed antitubercular activity against Mycobacterium tuberculosis H37Rv with MIC values of 72.7, 62.1, 9.1, 15.3, and 70.1 μM, respectively.
Keywords: Engelhardia roxburghiana; Juglandaceae; Stem; Diarylheptanoid; Naphthoquinone dimer; Tetralone; Antitubercular activity;

Sterols with antileishmanial activity isolated from the roots of Pentalinon andrieuxii by Li Pan; Claudio M. Lezama-Davila; Angelica P. Isaac-Marquez; Edward P. Calomeni; James R. Fuchs; Abhay R. Satoskar; A. Douglas Kinghorn (128-135).
A new cholesterol derivative, pentalinonsterol (1), and a new polyoxygenated pregnane sterol glycoside, pentalinonside (2), together with 18 known compounds, were isolated from the roots of the Mexican medicinal plant Pentalinon andrieuxii. Pentalinonsterol (1), and five known sterols (3, 4, 7, 15, 16) exhibited leishmanicidal activity against Leishmania mexicana in vitro. Morphological abnormalities and destruction of the amastigotes were observed by electron microscopy, as a result of treatment with compounds 1, 3, 4, 15 and 16.Display Omitted► Altogether 20 compounds were isolated and characterized from Pentalinon andrieuxii. ► Pentalinonsterol (1) and pentalinonside (2) are elucidated as hither to unknown sterol derivatives. ► Compound 1 and five known sterols were found to exhibit in vitro antileishmanial activity. ► This is the first time that sterols were reported as leishmanicidal principles of P. andrieuxii.A new cholesterol derivative, pentalinonsterol (cholest-4,20,24-trien-3-one, 1), and a new polyoxygenated pregnane sterol glycoside, pentalinonside (2), together with 18 known compounds, including 14 sterols (316), three coumarins (1719), and a triterpene (20), were isolated from a n-hexane partition of a methanol extract of the roots of the Mexican medicinal plant Pentalinon andrieuxii. Structure elucidation of compounds 1 and 2 was accomplished by spectroscopic data interpretation. All isolates were evaluated in vitro for their antileishmanial activity. Among these compounds, 6,7-dihydroneridienone (15) was found to be the most potent principle against promastigotes of Leishmania mexicana (L. mexicana). The cholesterol analogue, pentalinonsterol (1), together with two known sterols, 24-methylcholest-4,24(28)-dien-3-one (3) and neridienone (16), also exhibited significant leishmanicidal activity in this same bioassay. Compounds 1, 3, 15, 16, cholest-4-en-3-one (4), and cholest-5,20,24-trien-3β-ol (7), showed strong antileishmanial activity against amastigotes of L. mexicana, and 4 was found to be the most potent agent with an IC50 value of 0.03 μM. All the isolates were also evaluated for their cytotoxicity in non-infected bone marrow-derived macrophages, but none of these compounds was found active towards this cell line. The intracellular parasites treated with compounds 1, 3, 4, 15, and 16 were further studied by electron microscopy; morphological abnormalities and destruction of the amastigotes were observed, as a result of treatment with these compounds.
Keywords: Pentalinon andrieuxii (syn. Urechites andrieuxii); Apocynaceae; Sterols; Antileishmanial activity; Leishmania mexicana; Promastigotes; Amastigotes; Macrophages; Electron microscopy;

Prenylated flavonoids from Desmodium caudatum and evaluation of their anti-MRSA activity by Hisako Sasaki; Yoshiki Kashiwada; Hirofumi Shibata; Yoshihisa Takaishi (136-142).
Seven prenylated flavonoids (17) and a prenylated chromanochroman derivative (8), were isolated from the roots of Desmodium caudatum. The anti-bacterial activity of 12 compounds against methicillin-resistant Staphylococcus aureus was evaluated.Display Omitted► Seven flavonoids and a chromanochroman were isolated from Desmodium caudatum. ► The anti-MRSA activity of 12 compounds was evaluated. ► Some of the tested compounds showed relatively potent anti-MRSA activity. ► The presence of a prenyl or a 2,2-dimethylpyran group was suggested to be important.Seven prenylated flavonoids and a prenylated chromanochroman derivative, together with eight known flavonoids, were isolated from roots of Desmodium caudatum. The 15 structures were elucidated by extensive spectroscopic analyses. The antibacterial activity of many of other compounds was evaluated against methicillin-resistant Staphylococcus aureus (MRSA: COL and 5) by a disc diffusion method, and the minimum inhibitory concentrations (MICs) to MRSA were determined.
Keywords: Desmodium caudatum; Fabaceae; Prenylated flavonoid; Anti-methicillin-resistant Staphylococcus aureus (MRSA);

Co-occurrence of phenylphenalenones and flavonoids in Xiphidium caeruleum Aubl. flowers by Jingjing Fang; Dirk Hölscher; Bernd Schneider (143-148).
The natural product composition of Xiphidium caeruleum flowers was investigated using LC–DAD–SPE–NMR. Co-occurrence of phenylphenalenones and flavonoids in the Haemodoraceae was shown for the first time. Five natural products including four phenylbenzoisoquinolinones and a phenylisobenzochromenone were identified for the first time together with 17 known compounds.Display Omitted► The phytochemical profile of Xiphidium caeruleum flowers was studied by LC–SPE–NMR. ► The metabolites present in the flowers were found to differ to other plant parts. ► Five phenylphenalenone-type compounds were identified or the first time. ► First report of phenalenones and flavonoids co-occurring in the Haemodoraceae.A Xiphidium caeruleum flower extract was separated by semi-preparative HPLC into five fractions, from which three flavonoids, two phenylphenalenones and 17 phenylphenalenone-related compounds including five unknown compounds, were isolated and their structures elucidated by Liquid Chromatography–Diode Array Detection–Solid Phase Extraction–Nuclear Magnetic Resonance spectroscopy (LC–DAD–SPE–NMR) and mass spectrometry (MS). This is the first report of the co-occurrence of phenylphenalenones and flavonoids in the Haemodoraceae family. The ecological implications of flavonoids and various phenylphenalenone-type compounds and their putative biosynthesis sites in X. caeruleum are subject to discussion.
Keywords: Xiphidium caeruleum; Haemodoraceae; LC–DAD–SPE–NMR; Flavonoids; Phenylbenzoisochromenones; Phenylbenzoisoquinolinones; Phenylnaphthalic anhydrides; Phenylphenalenones;

Cyclic glycolipids from glandular trichome exudates of Cerastium glomeratum by Teigo Asai; Yuki Nakamura; Yui Hirayama; Kiyoshi Ohyama; Yoshinori Fujimoto (149-157).
Chemical investigation of glandular trichome exudates of Cerastium glomeratum (Caryophyllaceae) led to identification of fourteen 1,6′-cyclic ester derivatives of 9-, 10- and 11-glucopyranosyloxydocosanoic acids (glomerasides A–N).Fourteen cyclic glycolipids, named glomerasides A–N, have been isolated from the glandular trichome exudate of Cerastium glomeratum (Caryophyllaceae). Their structures were determined by spectroscopic analysis of the glycolipids, as well as by application of the Ohrui–Akasaka method to the fatty acid methyl esters derived from the glycolipids and GCMS studies of trimethylsilyl ether derivatives of the methyl esters. The various glomerasides have a glycosidic linkage between the anomeric hydroxy group of the glucose and the C-11, C-10 or C-9 positions of the docosanoyl moiety. They also contained an ester linkage between the C-6 hydroxy group of the glucose ring and the carboxyl group of the oxygenated fatty acid to form their macrocyclic structures. The glucose moiety was optionally acetylated and/or malonylated at the C-2 or C-3 hydroxy groups. Among these compounds, the 1,6′-cyclic ester of 11(R)-(2-O-acetyl-β-d-glucopyranosyloxy)docosanoic acid (glomeraside D) was the most abundant (25%).
Keywords: Cerastium glomeratum; Caryophyllaceae; Glandular trichome; Cyclic glycolipid; Ohrui–Akasaka method;

Sesquiterpenes from the conifer root rot pathogen Heterobasidion occidentale by David Hansson; Audrius Menkis; Kajsa Himmelstrand; Mattias Thelander; Åke Olson; Jan Stenlid; Magnus Karlsson; Anders Broberg (158-165).
Six sesquiterpenes were isolated and characterized from the basidiomycete Heterobasidion occidentale. The compounds were suggested to be intermediates in the biosynthesis of the sesquiterpene fomannosin.Display Omitted► Six sesquiterpenes were identified from the basidiomycete Heterobasidion occidentale. ► The sesquiterpenes were suggested to be involved in the biosynthesis of fomannosin. ► The biosynthesis was suggested to include one Baeyer–Villiger monooxygenase. ► Four of the sesquiterpenes were antifungal and phytotoxic and one was antibacterial.Investigation of the production of secondary metabolites of Heterobasidion occidentale led to the isolation and identification of six sesquiterpenes (illudolone A and B, illudolactone A and B, deoxyfomannosin A and B) along with the well-known sesquiterpene fomannosin and the previously described benzohydrofuran fomannoxin. The structures and relative configurations of the compounds were determined by 1D and 2D NMR spectroscopic analysis as well as by HRMS. Their absolute configuration and biosynthesis were suggested and discussed in relation to fomannosin. Four compounds showed growth inhibiting activity against several basidiomycetes, Phlebiopsis gigantea, Phanerochaete chrysosporium and H. occidentale, and toxicity towards the moss Physcomitrella patens. In addition, one compound displayed activity against the bacterium Variovorax paradoxus as well as against the ascomycete Fusarium oxysporum.
Keywords: Heterobasidion occidentale; Bondarzewiaceae; Secondary metabolite characterization; Sesquiterpenes; Fomannosin;

Phenolic alkaloids from the aerial parts of Dracocephalum heterophyllum by Limei Wang; Shuqi Wang; Shuang Yang; Xiaojiang Guo; Hongxiang Lou; Dongmei Ren (166-171).
Phenolic alkaloids, including flavonoidal alkaloids and an imidazole alkaloid, were obtained from Dracocephalum heterophyllum. The existence of flavonoidal alkaloids as racemates was demonstrated by HPLC using a chiral phase.Display Omitted► Phenolic alkaloids were isolated from Dracocephalum heterophyllum. ► The alkaloids, drahebephins A–B, are racemic. ► Racemates were resolved by HPLC using a chiral phase. ► CD spectroscopy was used for determination of absolute configurations.Chemical examination of aerial parts of Dracocephalum heterophyllum resulted in isolation of phenolic alkaloids, including two flavonoidal alkaloids, drahebephins A and B, one imidazole alkaloid with a phenolic substituent, drahebenine, together with 15 other known compounds. Their structures were established by extensive spectroscopic data analyses. Due to the stereogenic center in the pyrrolidin-2-one ring, the flavonoidal alkaloids are chiral, although they were isolated as racemates. The enantiomers were separated by HPLC using a chiral phase and stereochemically characterized by circular dichroism (CD) spectroscopy. The structure of compound drahebenine was established by X-ray crystallography.
Keywords: Dracocephalum heterophyllum; Lamiaceae; Flavonoidal alkaloid; Imidazole alkaloid; Chiral HPLC; Absolute configuration;

Phenylpropanoids from Phragmipedium calurum and their antiproliferative activity by Courtney M. Starks; Russell B. Williams; Vanessa L. Norman; Julie A. Lawrence; Mark O’Neil-Johnson; Gary R. Eldridge (172-175).
Seven stilbenes and one alkylresorcinol were isolated from the orchid Phragmipedium calurum during a screen for anticancer compounds. They were evaluated for antiproliferative activity against multiple human cancer cell lines. Two of these compounds displayed moderate activity against several cell lines.Display Omitted► Seven stilbenes and an alkylresorcinol were isolated from Phragmipedium calurum. ► These compounds were found during a screen for anticancer agents. ► Two of them displayed moderate antiproliferative activity.Seven stilbenes and one alkylresorcinol were isolated from the orchid Phragmipedium calurum during a screen for anticancer compounds. They were evaluated for antiproliferative activity against multiple human cancer cell lines, and two displayed moderate activity against several cell lines.
Keywords: Phragmipedium calurum; Orchidaceae; Stilbene; Alkylresorcinol; Cytotoxicity; Cancer;