Phytochemistry (v.65, #13)

Seasonal cardenolide production and Dop5 βr gene expression in natural populations of Digitalis obscura by Luis Roca-Pérez; Rafael Boluda; Isabel Gavidia; Pedro Pérez-Bermúdez (1869-1878).
Productivity variations and seasonal fluctuations of cardenolides have been studied in 10 natural populations of Digitalis obscura. Isolation and seasonal expression of the gene encoding the progesterone 5β-reductase are reported.Productivity variations and seasonal fluctuations of cardenolides have been studied in 10 natural populations of Digitalis obscura distributed in three bioclimatic belts. Main cardenolides in D. obscura plants are those of the series A and such predominance (ca. 80–85%) over the series B metabolites is independent of the population studied or the degree of maturity of the leaves. Primary glycosides represent ca. 50–60% of total cardenolides; this percentage did not vary among populations or with the leaf age but increased in summer and decreased in winter. A correlation analysis between plant biomass and cardenolide content showed a positive relationship of these parameters, which, according to the bioclimatic distribution of the populations, suggests that certain environmental conditions may cause marked decreases in plant biomass together with a reduction in productivity. Cardenolide contents changed in the timecourse of the four seasons as a multiple response to distinct plant and/or environmental factors. The lowest production was recorded in May, followed by a fast cardenolide accumulation in summer, a decreasing phase in autumn, and a stationary phase in winter. We also analysed the seasonal expression of the gene encoding the progesterone 5β-reductase, enzyme producing the required 5β-configured intermediaries of cardenolides. A fragment of the isolated partial genomic sequence was used as a probe for Northern analysis to study the seasonal gene expression in selected populations. The expression pattern showed increasing levels from February to July and a further reduction in autumn, although harmful climatic conditions seems to induce overexpression of this gene.
Keywords: Digitalis obscura; Scrophulariaceae; Secondary metabolism; Seasonal variability in natural environment; Cardenolides; Dop5 βr gene; Progesterone 5β-reductase;

The class III peroxidase multigenic family in rice and its evolution in land plants by Filippo Passardi; David Longet; Claude Penel; Christophe Dunand (1879-1893).
An extensive search was performed in rice genome in order to draw up the complete list of class III peroxidases present in this organism. Phylogenetic studies performed in rice and other plants identified a putative primitive peroxidase, which probably played a crucial role in apparition of land plants.Plant peroxidases (class III peroxidases, E.C. 1.11.1.7) are secreted glycoproteins known to be involved in the mechanism of cell elongation, in cell wall construction and differentiation, and in the defense against pathogens. They usually form large multigenic families in angiosperms. The recent completion of rice (Oryza sativa japonica c.v. Nipponbare) genome sequencing allowed drawing up the full inventory of the genes encoding class III peroxidases in this plant. We found 138 peroxidase genes distributed among the 12 rice chromosomes. In contrast to several other gene families studied so far, peroxidase genes are twice as numerous in rice as in Arabidopsis. This large number of genes results from various duplication events that were tentatively traced back using a phylogenetic tree based on the alignment of conserved amino acid sequences. We also searched for peroxidase encoding genes in the major phyla of plant kingdom. In addition to gymnosperms and angiosperms, sequences were found in liverworts, mosses and ferns, but not in unicellular green algae. Two rice and one Arabidopsis peroxidase genes appeared to be rather close to the only known sequence from the liverwort Marchantia polymorpha. The possible relationship of these peroxidases with the putative ancestor of peroxidase genes is discussed, as well as the connection between the development of the class III peroxidase multigenic family and the emergence of the first land plants.
Keywords: Arabidopsis thaliana; Rice; Multigenic family; Evolution; Duplication; Laccase; Phytochrome; Peroxidase ancestor;

The sesquiterpene hydrocarbons of maize (Zea mays) form five groups with distinct developmental and organ-specific distributions by Tobias G. Köllner; Christiane Schnee; Jonathan Gershenzon; Jörg Degenhardt (1895-1902).
Analysis of the sesquiterpene content of young and mature maize plants revealed five groups of hydrocarbons, each with a different spatial and temporal distribution in the plant. Within each group, compounds co-occurred in the same constant ratio to one another, indicative of possible biosynthetic relationships.The sesquiterpene hydrocarbon chemistry of maize (Zea mays) inbred line B73 was analyzed by both direct solvent extraction and headspace sampling. In seedlings, 15 olefinic compounds were identified, and 21 olefins were detected in mature plants after anthesis. Both solvent extracts and collections of headspace terpenes were found to contain the same compounds in the same relative proportions suggesting that there is no selective barrier to release from plants. Approximately 25% of the stored pool was found to be released from young seedlings per hour. The individual sesquiterpenes varied extensively in their abundance among different organs and developmental stages. Compounds could be divided into five different groups such that the members of each group always occur together in the same constant ratios to one another. Each group has a distinct distribution pattern. Group A includes the two dominant compounds, (E)-β-farnesene and α-bergamotene, and appears only after herbivore damage in seedlings, but is constitutively present in the leaves and husks after anthesis. The major compounds of group B, including α-copaene, germacrene D and δ-cadinene, were present throughout the seedling but found only in husks of mature plants. The group C compounds, β-bisabolene and an unknown sesquiterpene olefin, are restricted to the roots. The presence of group D and E compounds was confined to the leaves and husk of mature plants. The complex sesquiterpene mixture of group D is identical to the products formed by the previously identified terpene synthase TPS4, suggesting that each of the four other sesquiterpene hydrocarbon mixtures may also represent the products of a single terpene synthase.
Keywords: Zea mays L.; Poaceae; Maize; Sesquiterpenes; Volatiles; Terpene biosynthesis; Terpene synthase; Volatile emission; Herbivore-induced terpene formation;

Changes in glycosidase activities during galactoglucomannan oligosaccharide inhibition of auxin induced growth by Ladislav Bilisics; Jozef Vojtaššák; Peter Capek; Karin Kollárová; Desana Lišková (1903-1909).
The inhibition of 2,4-D-induced elongation growth by galactoglucomannan oligosaccharides in pea stem segments (Pisum sativum L. cv. Tyrkys) after 18 h of incubation results in changes of extracellular, intracellular and cell wall glycosidase activities (β-d-glucosidase, β-d-mannosidase, β-d-galactosidase, β-d-xylosidase, α-d-galactosidase, and α-l-arabinosidase).The inhibition of 2,4-D-induced elongation growth by galactoglucomannan oligosaccharides (GGMOs) in pea stem segments (Pisum sativum L. cv. Tyrkys) after 18 h of incubation results in changes of extracellular, intracellular and cell wall glycosidase activities (β-d-glucosidase, β-d-mannosidase, β-d-galactosidase, β-d-xylosidase, α-d-galactosidase, and α-l-arabinosidase). GGMOs lowered the glycosidase activities in the extracellular fraction, while in the cell wall fractions their activities were markedly increased. The intracellular enzyme α-d-galactosidase increased while the β-d-galactosidase decreased in activity in response to the GGMO treatment. Extracellular enzymes showed low values of activities in comparison with intracellular and cell wall glycosidases. It is evident that GGMOs can alter auxin induced elongation and glycosidase activities in different compartments of the cell, however, the mode and site of their action remains unclear.
Keywords: Pisum sativum L. cv. Tyrkys; Fabaceae; Elongation growth; Glycosidases; Galactoglucomannan oligosaccharides;

Salt-induced lipid changes in Catharanthus roseus cultured cell suspensions by Salem Elkahoui; Abderrazek Smaoui; Mokhtar Zarrouk; Rachid Ghrir; Férid Limam (1911-1917).
Exposure of Catharanthus roseus cell suspensions to increasing salinity induced several changes in membrane lipid content and composition. The observed increase in phospholipid, free sterol and fatty acid unsaturation, suggests a modification in membrane fluidity.Salt treatment strongly affected cell growth by decreasing dry weight. Exposure of Catharanthus roseus cell suspensions to increasing salinity significantly enhanced total lipid (TL) content. The observed increase is mainly due to high level of phospholipids (PL). Hundred mM NaCl treatment increased phospholipid species phosphatidylcholine (PC) and phosphatidylethanolamine (PE), whereas it reduced glycolipid ones monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) but not sulfoquinovosyldiacylglycerol (SQDG). Moreover, fatty acid composition was clearly modified when cells were cultured in the presence of 100 mM NaCl, whereas only few changes occurred at 50 mM. Salt treatment decreased palmitic acid (16:0) level and increased that of linolenic acid (18:2). Such effect was observed in phospholipid species PC and PE and in glycolipid DGDG. Double bond index (DBI) was enhanced more than 2-fold in fatty acids of either glycolipids or phospholipids from cells submitted to 100 mM NaCl. Free sterol content was also significantly enhanced, especially at 100 mM NaCl, whereas free sterols/phospholipids (St/PL) ratio was slightly decreased. All these salt-induced changes in membrane lipids suggest an increase in membrane fluidity of C. roseus cells.
Keywords: Catharanthus roseus; Salt stress; Lipids; Fatty acids; Free sterols;

Isolation and characterization of wound-inducible carboxypeptidase inhibitor from tomato leaves by Monica Dı́ez-Dı́az; Vicente Conejero; Ismael Rodrigo; Gregory Pearce; Clarence A. Ryan (1919-1924).
Carboxypeptidase inhibitor protein, previously reported not to be wound-inducible in tomato leaves, was identified in wounded tomato leaves and was isolated and characterized.In a previous report [Mol. Gen. Genet. 228 (1991) 281], carboxypeptidase inhibitor protein (CPI) mRNA was found to accumulate in leaves of wounded tomato plants, but CPI protein could not be detected. In contrast, we found that CPI protein does accumulate in tomato leaves in response to wounding, and also in response to treatment with either systemin, methyl jasmonate (MeJ), oligogalacturonic acid, or chitosan. Identification of CPI protein was confirmed by its inhibition of metallo-carboxypeptidase A (CPAase), which was used as an assay during purification of the inhibitor from leaves of MeJ-treated tomato plants. Amino acid sequence analysis and mass spectroscopic analyses of the pure protein confirmed its identity as CPI. The pure protein inhibited CPAase in a 1:1 stoichimetric interaction. Time course analyses of the induction of CPI mRNA in tomato leaves in response to wounding indicated that the gene is a member of the group of “late genes” that code for defensive proteins synthesized in leaves in response to herbivore attack.
Keywords: Lycopersicon esculentum; cv Castlemart; Solanaceae; Tomato; Protein chemistry; Metallo-carboxypeptidase inhibitor;

Flavonoids from shoots and roots of Trifolium repens (white clover) grown in presence or absence of the arbuscular mycorrhizal fungus Glomus intraradices by Marı́a A. Ponce; José M. Scervino; Rosa Erra-Balsells; Juan A. Ocampo; Alicia M. Godeas (1925-1930).
Analysis of extracts obtained from Trifolium repens roots and shoots revealed that compositions of the flavonoid mixtures varied in the presence or absence of the arbuscular fungus Glomus intraradices.White clover (Trifolium repens) plants were grown in the presence or absence of the arbuscular mycorrhizal fungus Glomus intraradices. Flavones, 4,5,6,7,8-pentahydroxy-3-methoxyflavone and 5,6,7,8-tetrahydroxy-3-methoxyflavone, as well as two flavones 3,7-dihydroxy-4-methoxyflavone and 5,6,7,8-tetrahydroxy-4-methoxyflavone never previously reported in plants, were isolated. The known 3,5,6,7,8-pentahydroxy-4-methoxyflavone, 2,3,4,5,6-pentahydroxy-chalcone, 6-hydroxykaempferol, 4,5,6,7,8-pentahydroxyflavone and 3,4-dimethoxykaempferol were also obtained. Analysis of extracts obtained from roots and shoots revealed that the compositions of the flavonoid mixtures varied with growing conditions. Quercetin, acacetin and rhamnetin accumulated in roots of inoculated plants, whereas they were not detected in non-inoculated plants.
Keywords: White clover; Trifolium repens; Leguminosae; Shoots; Roots; Flavonoids; Flavones; Chalcone; Glomus intraradices; 4,5,6,7,8-Pentahydroxy-3-methoxyflavone; 5,6,7,8-Tetrahydroxy-3-methoxyflavone; 3,7-Dihydroxy-4-methoxyflavone; 5,6,7,8-Tetrahydroxy-4-methoxyflavone;

Mulinane-type diterpenoids from Azorella compacta display antiplasmodial activity by Luis A. Loyola; Jorge Bórquez; Glauco Morales; Aurelio San-Martı́n; Jose Darias; Ninoska Flores; Alberto Giménez (1931-1935).
Two mulinane-type diterpenoids were isolated from Azorella compacta: 20-hydroxymulin-11,13-dienyl acetate 1 and 13,14-dihydroxymulin-11-en-20-oic acid 2. Both compounds, as well as three previously isolated diterpenes were evaluated for new potential as in vivo growth inhibitors of Plasmodium berghei.Two mulinane-type diterpenoids were isolated from Azorella compacta; namely 20-hydroxymulin-11,13-dienyl acetate and 13,14-dihydroxymulin-11-en-20-oic acid. The structures were elucidated by analysis of their spectroscopic data. These compounds, as well as three previously isolated diterpenes, were evaluated as potential in vivo growth inhibitors of Plasmodium berghei NK 65 on infected mice at an intraperitoneal dose of 10 mg/kg/day. Sixty percent and forty-two percent growth inhibition were obtained with 17-acetoxymulin-11,13-dien-20-oic acid and 13, 14-dihydroxymulin-11-en-20-oic acid, respectively.
Keywords: Azorella compacta; Umbelliferae; Diterpenoids; Plasmodium berghei;

Fresh organically grown ginger (Zingiber officinale): composition and effects on LPS-induced PGE2 production by Shivanand D. Jolad; R.Clark Lantz; Aniko M. Solyom; Guan Jie Chen; Robert B. Bates; Barbara N. Timmermann (1937-1954).
Ginger extracts were analyzed directly by GC–MS without modification to trimethylsilyl ether derivatives. From organically grown Hawaiian white and yellow ginger varieties 51 compounds were identified, of which 20 had not been previously reported as ginger constituents.Gas chromatography in conjunction with mass spectrometry, a technique previously employed to analyze non-volatile pungent components of ginger extracts modified to trimethylsilyl derivatives, was applied successfully for the first time to analyze unmodified partially purified fractions from the dichloromethane extracts of organically grown samples of fresh Chinese white and Japanese yellow varieties of ginger, Zingiber officinale Roscoe (Zingiberaceae). This analysis resulted in the detection of 20 hitherto unknown natural products and 31 compounds previously reported as ginger constituents. These include paradols, dihydroparadols, gingerols, acetyl derivatives of gingerols, shogaols, 3-dihydroshogaols, gingerdiols, mono- and diacetyl derivatives of gingerdiols, 1-dehydrogingerdiones, diarylheptanoids, and methyl ether derivatives of some of these compounds. The thermal degradation of gingerols to gingerone, shogaols, and related compounds was demonstrated. The major constituent in the two varieties was [6]-gingerol, a chemical marker for Z. officinale. Mass spectral fragmentation patterns for all the compounds are described and interpreted. Anti-inflammatory activities of silica gel chromatography fractions were tested using an in vitro PGE2 assay. Most of the fractions containing gingerols and/or gingerol derivatives showed excellent inhibition of LPS-induced PGE2 production.
Keywords: Zingiber officinale; Zingiberaceae; Ginger; Rhizomes; Ginger derivatives;

Minor pyrano-isoflavones from Eriosema kraussianum: activity-, structure-, and chemical reaction studies by Siegfried E. Drewes; Marion M. Horn; Fatima Khan; Orde Q. Munro; Jabu T.B. Dhlamini; Christopher Rakuambo; J.J. Marion Meyer (1955-1961).
The structure of one of the minor isoflavones is shown (7). X-ray studies confirm the structure of the pyrano-isoflavone isolated previously.The isolation and identification of two minor pyrano-isoflavones from Eriosema kraussianum is described. New studies on the original pyrano-isoflavones shows that: (i) kraussianone 2 (a major compound in the plant) can be cyclised under acid conditions, (ii) kraussianones 3 and 5 cause contraction (not relaxation as anticipated) of corpus cavernosum tissue and (iii) the structures proposed previously for 4 and 5 are confirmed by the data obtained from an X-ray study of 5.
Keywords: Eriosema kraussianum; Pyrano-isoflavones; Cyclization; Smooth muscle contraction; X-ray;

Insect growth inhibition by tocotrienols and hydroquinones from Roldana barba-johannis by Carlos L. Céspedes; Patricio Torres; Juan C. Marı́n; Amira Arciniegas; Alfonso Romo de Vivar; Ana L. Pérez-Castorena; Eduardo Aranda (1963-1975).
Tocotrienols and hydroquinones displaced insecticidal and insect growth inhibitory activities against Fall Armyworm (Spodoptera frugiperda).The methanol extract from the aerial parts of Roldana barba-johannis (Asteraceae) afforded sargachromenol, sargahydroquinoic acid, and sargaquinoic acid. These natural products and their corresponding acetylated and methylated derivatives showed insecticidal and insect growth regulatory activities against the Fall Armyworm [Spodoptera frugiperda J.E. Smith, (Lepidoptera: Noctuidae)], an important insect pest of corn. The most active compounds were sargachromenol and its acetylated derivative; sargahydroquinoic acid and its acetylated derivative; and a mixture of sargachromenol, sargahydroquinoic acid, and sargaquinoic acid (6:3:1) and the acetylated form of this mixture. All these compounds and mixtures had significant inhibitory effects between 5.0 and 20.0 ppm in diets. Most compounds were insecticidal to larvae, with lethal doses between 20 and 35 ppm. In addition, these substances also demonstrated scavenging properties toward 2,2-diphenyl-1-picrylhydrazyl radical in TLC autographic and spectrophotometric assays. These compounds appear to have selective effects on the pre-emergence metabolism of the insect. The results from these compounds were fully comparable in activity to those known natural insect growth inhibitors such as gedunin and methanol extracts of Cedrela salvadorensis and Yucca periculosa. These substances may be useful as natural insecticidal agents.
Keywords: Roldana barba-johannis; Asteraceae; Tocotrienols; Hydroquinones; Insect-growth regulation; Spodoptera frugiperda; Fall Armyworm;

Antioxidant phenolic and quinonemethide triterpenes from Cheiloclinium cognatum by Alex Haroldo Jeller; Dulce Helena Siqueira Silva; Luciano Morais Lião; Vanderlan da Silva Bolzani; Maysa Furlan (1977-1982).
The triterpenes, 22β-hydroxypristimerin and cognatine, were isolated together with seven known compounds from Cheiloclinium cognatum. The isolates were investigated for their radical scavenging abilities.The triterpenes, 22β-hydroxypristimerin and cognatine, were isolated together with the known compounds pristimerin, maytenin, 20α-hydroxymaytenin, 22β-hydroxymaytenin, netzahualcoyol, netzahualcoyondiol and netzahualcoyone from root bark of Cheiloclinium cognatum. The structures of the isolated compounds were elucidated by interpretation of their spectral data, including gHMQC and gHMBC experiments.The isolates were investigated for their radical scavenging abilities through a spectrophotometric assay involving reduction of 2,2-diphenyl-picryl hydrazyl (DPPH).
Keywords: Cheiloclinium cognatum; Hippocrateaceae; Quinonemethide triterpenes; Phenolic triterpene; Antioxidant activity;

Immunomodulatory study revealed the capacity of all ion-exchange fractions of the polysaccharide complex A to induce the proliferation of rat thymocytes in the order A2  > A3=A4  > A1=A5=A6  > A.A water-soluble polysaccharide complex (A) composed of galactose (17.9%), 3-O-methyl-galactose (3.0%), glucose (15.5%), mannose (8.3%), arabinose (30.4%), xylose (7.6%), fucose (2.6%), rhamnose (6.7%), and uronic acids (8.0%) has been isolated from the aerial parts of sage (Salvia officinalis L.) by cold water extraction. It showed a broad molecular-mass distribution pattern (M w ∼2000–93 000) with a predominance of polymers with M w <10 000. Ion-exchange chromatography of A afforded six polymeric fractions (A1–A6) in which arabinogalactans associated with galacturonan and/or rhamnogalacturonan backbones prevail. Sage polysaccharides were examined for their immunomodulatory activity in the comitogenic thymocyte test which is interpreted as being an in vitro correlate of adjuvant activity. The acidic polysaccharide fractions A2, A3 and A4 exhibited the highest mitogenic and comitogenic activities of all fractions tested, and relatively high SIcomit/SImit ratios ∼3 indicate potential adjuvant properties of these polysaccharides.
Keywords: Sage; Salvia officinalis; Arabinogalactans; Rhamnogalacturonans; FT-IR and NMR spectroscopies; Immunostimulatory activity;

Metabolomic analysis of Strychnos nux-vomica, Strychnos icaja and Strychnos ignatii extracts by 1H nuclear magnetic resonance spectrometry and multivariate analysis techniques by Michel Frédérich; Young Hae Choi; Luc Angenot; Goetz Harnischfeger; Alfons W.M. Lefeber; Robert Verpoorte (1993-2001).
1H Nuclear magnetic resonance spectrometry and multivariate analysis techniques were applied for the metabolic profiling of three Strychnos species: Strychnos nux-vomica, Strychnos ignatii, and Strychnos icaja. 1H Nuclear magnetic resonance spectrometry and multivariate analysis techniques were applied for the metabolic profiling of three Strychnos species: Strychnos nux-vomica (seeds, stem bark, root bark), Strychnos ignatii (seeds), and Strychnos icaja (leaves, stem bark, root bark, collar bark). The principal component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between all samples, using the three first components. The key compounds responsible for the discrimination were brucine, loganin, fatty acids, and Strychnos icaja alkaloids such as icajine and sungucine. The method was then applied to the classification of several “false angostura” samples. These samples were, as expected, identified as S. nux-vomica by PCA, but could not be clearly discriminated as root bark or stem bark samples after further statistical analysis.
Keywords: Loganiaceae; Strychnos nux-vomica; Strychnos ignatii; Strychnos icaja; Strychnine; Brucine; Metabolic profiling; 1H NMR; Multivariate analysis; PCA; Alkaloid;

Constituents from the bark of Tabebuia impetiginosa by Tsutomu Warashina; Yoshimi Nagatani; Tadataka Noro (2003-2011).
Sixteen compounds were isolated from the bark of Tabebuia impetiginosa, whose structures were determined using both spectroscopic and chemical methods.The bark of Tabebuia impetiginosa afforded nineteen glycosides, consisting of four iridoid glycosides, two lignan glycosides, two isocoumarin glycosides, three phenylethanoid glycosides and eight phenolic glycosides. Their structures were determined using both spectroscopic and chemical methods. Iridoid glycosides, phenylethanoid glycosides and lignan glycosides had ajugol, osmanthuside H and secoisolariciresinol 4-O-β-d-glucopyranoside as their structural elements, respectively, whereas the aglycone moieties of the isocoumarin glycosides were considered to be (−)-6-hydroxymellein. Phenolic glycosides had 4-methoxyphenol, 2,4-dimethoxyphenol, 3,4-dimethoxyphenol, 3,4,5-trimethoxyphenol and vanillyl 4-hydroxybenzoate as each aglycone moiety. Additionally, the sugar chains of these isocoumarin glycosides and phenolic glycosides were concluded to be β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranoside as well as those of osmanthuside H and above phenylethanoid glycosides.
Keywords: Tabebuia impetiginosa; Bignoniaceae; Iridoid; Lignan; Isocoumarin; Phenylethanoid; Phenolic glycoside;

Erratum to: “Further saponins from Meryta lanceolata” [Phytochemistry 65 (2004) 909–914] by F.R. Melek; Toshio Miyase; N.S. Ghaly; M.F. Yousif (2015).