Phytochemistry (v.66, #5)

Author index (III).

Identification of an EcoRI restriction site for a rapid and precise determination of β-asarone-free Acorus calamus cytotypes by Cinzia M. Bertea; Chiara M.M. Azzolin; Simone Bossi; Giovanni Doglia; Massimo E. Maffei (507-514).
The aim of this work was to identify a diploid β-asarone-free Acorus calamus by using chemical and molecular approaches. A. calamus was analyzed by gas chromatography–mass spectrometry (GC–MS) and comparison of the 700 bp sequence of a 5S-rRNA gene spacer region was performed. By aligning the isolated nucleotide sequences of varieties of A. calamus chemotypes present in Genbank, sequence diversities were found in the spacer region. The PCR products were digested by using EcoRI. The restriction profile of the spacer domain resulted different for the two cytotypes.Calamus (Acorus calamus L., Araceae) is an aromatic herb, indigenous to Central Asia and Eastern Europe. The fragrant oils obtained by alcoholic extraction of the rhizome are mainly used in the pharmaceutical and oenological industries. Nevertheless, the occurrence of β-asarone [(Z)-1,2,4-trimethoxy-5-prop-1-enyl-benzene] limits the possibility of its use due to the carcinogenic properties of this compound.The aim of this work was to identify a diploid β-asarone-free A. calamus by using chemical and molecular approaches. For these purposes alcoholic extracts of both diploid and triploid A. calamus were analyzed by gas chromatography–mass spectrometry (GC–MS) and comparison of the 700 bp sequence of the non-transcribed spacer (NTS) in the 5S-rRNA gene was also performed.Alcoholic extracts of the triploid A. calamus were characterized by a higher percentage of β-asarone (11%), which was the main compound, followed by higher percentages of camphene (2.27%), E-β-ocimene (3.28%), camphor (1.54%), calarene (1.42%), α-selinene (5.02%) and τ-cadinol (2.00%), when compared to the diploid A. calamus. The latter had higher percentages of iso-shyobunone (8.62%), β-sesquiphellandrene (3.28%), preiso calamendiol (22.81%) and acorone (26.33%), and completely lacked of β-asarone.The 5S-rRNA spacer region of both diploid and triploid A. calamus were amplified by PCR using a pair of primers located at the 3′ and 5′ ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 700 bp) were gel purified, subcloned into pGEM®-T Easy vector and sequenced. By aligning the isolated nucleotide sequences of the two varieties and the sequences from different A. calamus chemotypes present in Genbank, sequence diversities were found in the spacer region. Furthermore, the PCR products were digested by using EcoRI. The restriction profile of the spacer domain resulted different for the two cytotypes.Along with chemical analysis of alcoholic extracts, sequence analysis coupled to restriction mapping was demonstrated to represent a powerful tool to distinguish the A. calamus diploid cytotype from the others. The security and effective usage of the diploid β-asarone-free A. calamus was also discussed.
Keywords: Acorus calamus L.; Araceae; Sweetflag; Oil chemical composition; β-Asarone; Diploid; Triploid; 5S-rRNA spacer region; DNA sequence analysis; EcoRI site restriction mapping;

Purification and cloning of a γ-glutamyl transpeptidase from onion (Allium cepa) by Martin L. Shaw; Meeghan D. Pither-Joyce; John A. McCallum (515-522).
Onion γ-glutamyl transpeptidase catalyses hydrolysis of γ-glutamyl linkages in γ-glutamyl peptides and transfer of the γ-glutamyl group to amino acids and peptides and has high affinity for glutathione and glutathione conjugates.γ-Glutamyl transpeptidase (E.C. 2.3.2.2; GGT) catalyses hydrolysis of γ-glutamyl linkages in γ-glutamyl peptides and transfer of the γ-glutamyl group to amino acids and peptides. Although plant γ-glutamyl peptide metabolism is important in biosynthesis and metabolism of secondary products and xenobiotics, plant GGTs are poorly characterised. We purified a membrane-associated GGT from sprouting onion bulbs that catalyses transpeptidation of methionine by the synthetic substrate γ-glutamyl-p-nitroanilide (GGPNA) and obtained N-terminal peptide sequence. We also cloned the full-length coding region of an onion GGT by homology with the Arabidopsis enzyme and confirmed that this shared the same N-terminal sequence. Enzyme kinetic studies show that the enzyme has high affinity for glutathione and glutathione conjugates, and that affinity for S-substituted glutathione analogs decreases as the substituted chain length increases. The major onion γ-glutamyl peptide, γ-glutamyl trans-S-1-propenyl cysteine sulfoxide (GGPrCSO) exhibited uncompetitive inhibition of transpeptidation by GGPNA. This suggests that GGPrCSO is a poor glutamyl donor and therefore unlikely to be an in vivo substrate for peptidase activity by this enzyme.
Keywords: Allium cepa; Alliaceae; Onion; Enzymology; Cloning; γ-Glutamyl peptides; γ-Glutamyl transpeptidase; Glutathione;

Analysis of polyamine metabolism in soybean seedlings using 15N-labelled putrescine by Masato Ohe; Masaki Kobayashi; Masaru Niitsu; Nello Bagni; Shigeru Matsuzaki (523-528).
Translocation and metabolism during soybean germination using 15N-labelled putrescine is reported.The translocation and metabolism of polyamines during soybean germination were studied using 15N-labelled putrescine as a precursor. Both 15N-labelled and unlabelled polyamines were simultaneously detected using a novel application of ionspray ionization-mass spectrometry. 15N-putrescine was rapidly transported to the shoots and roots, where it was converted to spermidine and spermine. The main 15N-polyamine that accumulated in the root was 15N-spermine. It was found that there were differences in the way endogenous putrescine and exogenous 15N-putrescine were metabolized in soybean seedlings.
Keywords: Glycine max; Leguminosae; Soybean; Metabolism; Translocation; Polyamine;

Mass spectral characterization of fatty acid amides from alfalfa trichomes and their deterrence against the potato leafhopper by Christopher M. Ranger; Rudolph E.K. Winter; George E. Rottinghaus; Elaine A. Backus; David W. Johnson (529-541).
N-(3-methylbutyl)amides and N-(2-methylpropyl)amides of C14 through C18 fatty acids were identified from glandular trichomes of the alfalfa genotype Medicago sativa G98A., and synthetic representatives of the amides were tested for bioactivity against the potato leafhopper, Empoasca fabae.A homologous series of N-(3-methylbutyl)amides of normal saturated C14, C15, C16, C17 and C18 fatty acids were identified as major components of glandular trichome extracts from Medicago sativa G98A, an alfalfa genotype resistant to the potato leafhopper, Empoasca fabae. A second homologous series of N-(2-methylpropyl)amides of C14 through C18 normal fatty acids were minor components. Saturated free fatty acids C12, C13, C14, C15, C16, C17 and C18 were present in trace amounts, as was the N-(3-methylbutyl)amide of linoleic acid (C18:2). N-(3-methylbutyl)amides and N-(2-methylpropyl)amides of C14 through C18 fatty acids, along with the N-(3-methylbutyl)amide of linoleic acid, were synthesized and bioassayed for leafhopper deterrence by applying the compounds to the surface of a sachet containing an artificial diet. Leafhoppers were then offered a two-way choice between diet surfaces treated with the synthetic amides or an untreated control. N-(3-methylbutyl)amides and N-(2-methylpropyl)amides of C14 through C18 fatty acids did not deter leafhopper settling in a dose-dependent fashion. In contrast, when tested singly, N-(3-methylbutyl)amide of linoleic acid exhibited dose-dependent deterrence against leafhopper settling. Fatty acid amides localized in alfalfa glandular trichomes likely contribute to leafhopper resistance.
Keywords: Alfalfa; Medicago sativa; Leguminosae; Papilionaceae; Insect resistance; Fatty acid amides;

Leucine aminopeptidase M inhibitors, cyanostatin A and B, isolated from cyanobacterial water blooms in Scotland by Tomoharu Sano; Hiroo Takagi; Louise F. Morrison; James S. Metcalf; Geoffrey A. Codd; Kunimitsu Kaya (543-548).
Two leucine aminopeptidase M inhibitors, cyanostatin A and B, were isolated from water blooms of Loch Rescobie in Scotland. These inhibitors were lipopeptides contained 3-amino-2-hydroxydecanoic acids. Cyanostatin A and B strongly inhibited the activity of leucine aminopeptidase M at IC50 values of 40 and 12 ng/ml, respectively.Two leucine aminopeptidase M inhibitors, cyanostatin A and B, were isolated from cyanobacterial water blooms at Loch Rescobie in Scotland, and specifically from a Microcystis species. Both inhibitors were lipopeptides containing 3-amino-2-hydroxydecanoic acid and weak inhibitors of protein phosphatase (PP2A). Both strongly inhibited the activity of leucine aminopeptidase M with IC50 values of 40 and 12 ng/ml, respectively.
Keywords: Cyanobacteria; Microcystis sp., Lipopeptide; Leucine aminopeptidase M inhibitor; Water bloom; Microginin; 3-Amino-2-hydroxydecanoic acid;

Chemotaxonomy of the Rubiaceae family based on leaf fatty acid composition by Sébastien Mongrand; Alain Badoc; Brigitte Patouille; Chantal Lacomblez; Marie Chavent; Jean-Jacques Bessoule (549-559).
107 Rubiaceae leaves were analyzed for their fatty acid composition. One of the most interesting variables was all cis7,10,13-16:3 hexadecatrienoic acid. With the exception of Rubieae, Theligoneae, and some Anthospermeae which are typically 16:3-plants, other Rubiaceae appeared to be C18:3 plants.With 10,700 species distributed in 637 genera, the Rubiaceae family is one of the largest of the angiosperms. Since it was previously evidenced that the fatty acid composition of photosynthetic tissues can be a tool for chemotaxonomic studies, the fatty acid composition of leaves from 107 Rubiaceae species highly representative of the diversity of the family was determined. Principal component analysis allowed a clear-cut separation of Coffeae, Psychotrieae and Rubieae. The occurrence of C16:3 fatty acid, a marker of the prokaryotic plastidial lipid biosynthetic pathway, concerned at least two branches: Theligoneae/Rubieae and Anthospermeae–Anthosperminae which appeared to be in close relationship. Additional experiments were carried out to ensure the correlation between the presence of C16:3 fatty acid and the prokaryotic biosynthetic pathway.
Keywords: Rubiaceae; Chemotaxonomy; Leaf fatty acids; Galactolipids; All cis7,10,13-hexadecatrienoic acid; Anteiso 17:0 fatty acid; Multivariate analysis;

Genetic diversity of UPASI tea clones (Camellia sinensis (L.) O. Kuntze) on the basis of total catechins and their fractions by M. Saravanan; K.M. Maria John; R. Raj Kumar; P.K. Pius; R. Sasikumar (561-565).
Group 1 is a mixture of “Assam” and “China” cultivars, including medium quality and drought tolerant clones. Group 2 contained purely “China” cultivars, while group 3 possessed high quality tea cultivars. Group 4 contained both “Assam” and “China” cultivars, while group 5 comprised only “Assam” cultivars.Tea leaf catechins and the ratio of dihydroxylated to trihydroxylated catechin fractions were analysed to identify the genetic diversity of 26 UPASI released tea clones. Principal component analysis (PCA) based on regression factor separated tea clones into five groups according to their jats (Jats are region based rays for e.g., Assam, China and Cambod origin) as well as their quality constituents (such as total polyphenols, total catechins, amino acids in the green leaves and liquor characteristics of black tea), particularly the catechins. Group 1 represented medium quality (quality of the final produce) clones, such as UPASI-10, UPASI-12 and UPASI-15 and drought tolerant clones like UPASI-1, UPASI-2, UPASI-9 and UPASI-10. Group 2 contained purely “China” cultivars while group 3 possessed high quality tea cultivars. “Assam” (group 5) teas had the lowest ratio of dihydroxylated to trihydroxylated catechin fractions (1:4) than the “Chinery” (group 2) teas (1:5). This biochemical differentiation indicated that there is a vast genetic diversity in UPASI released tea clones in terms of catechin fractions, even though the majority of them were selected from one tea estate located in the Nilgiris.
Keywords: Genetic diversity; Total catechins and catechin fractions;

Chemical constituents of Murraya siamensis: three coumarins and their anti-tumor promoting effect by Chihiro Ito; Masataka Itoigawa; Saori Onoda; Atsuko Hosokawa; Nijsiri Ruangrungsi; Toshimitsu Okuda; Harukuni Tokuda; Hoyoku Nishino; Hiroshi Furukawa (567-572).
Three coumarins, named murrayacoumarins A (1), B (2), and C, were isolated from the leaves of Murraya siamensis as inhibitors of Epstein–Barr virus early antigen activation induced by 12-O-tetradecanoylphorbol-13-acetate in Raji cells, along with eight known compounds. Their structures were elucidated on the basis of spectroscopic analyses.Isolation and structure elucidation of three coumarins, murrayacoumarins A, B, and C, together with eight known coumarins, from the leaves of Murraya siamensis Craib collected in Thailand are described. Results of a primary screening of inhibitory effects of seven of these compounds on 12-O-tetradecanoylphorbol-13-acetate-induced Epstein–Barr virus early antigen activation in Raji cells are also presented.
Keywords: Murraya siamensis; Rutaceae; Coumarins; Anti-tumor promoting effect; Epstein–Barr virus activation test;

Chromenes of polyketide origin from Peperomia villipetiola by Karina J. Malquichagua Salazar; Guillermo E. Delgado Paredes; Luis Ripalda Lluncor; Maria Claudia Max Young; Massuo Jorge Kato (573-579).
The extracts of leaves and stems of Peperomia villipetiola have been found to contain myristicin and seven chromenes (17) of orsellinic acid-type. The anti-fungal activities of the chromenes measured by bioautography against Cladosporium cladosporioides and Cladosporium sphaerospermum indicated compounds 6 and 7 as the most active.An extract of leaves and stems of Peperomia villipetiola has been found to contain myristicin (3-methoxy-4,5-methylenedioxy-allylbenzene) and seven chromenes, whose structures are methyl 5-hydroxy-7-methyl-2,2-dimethyl-2H-1-chromene-6-carboxylate (1), methyl 5-methoxy-7-methyl-2,2-dimethyl-2H-1-chromene-8-carboxylate (2), methyl 7-hydroxy-5-methyl-2,2-dimethyl-2H-1-chromene-6-carboxylate (3), methyl 7-methoxy-5-methyl-2,2-dimethyl-2H-1-chromene-6-carboxylate (4), 5-methanol-7-hydroxy-2,2-dimethyl-2H-1-chromene-6-carboxylic acid (5), 5-methanol-7-methoxy-2,2-dimethyl-2H-1-chromene-6-carboxylic acid (6), and methyl 5-acetoxymethanol-7-hydroxy-2,2-dimethyl-2H-1-chromene-6-carboxylate (7). A biosynthetic rationale for 17 suggests that orsellinic acid may be a common intermediate. The anti-fungal activities of the chromenes were measured bioautographically against Cladosporium cladosporioides and Cladosporium sphaerospermum: compounds 6 and 7 were found to be the most active.
Keywords: Peperomia villipetiola; Piperaceae; Structural elucidation; Anti-fungal activity; Chromenes; Orsellinic acid;

Synthesis and bioactivity of C-29 brassinosteroid analogues with different functional groups at C-6 by Javier A. Ramírez; Carme Brosa; Lydia R. Galagovsky (581-587).
The synthesis and bioactivity of four synthetic analogues of 28-homobrassinosteroids bearing different functional groups at C-6 is described.In this paper, we report the synthesis and bioactivity of four synthetic analogues of 28-homobrassinosteroids, in order to evaluate the influence in bioactivity when the C-6 keto group is replaced by different functional groups. The synthetic analogues are 6-deoxo-28-homocastasterone [(22R,23R)-stigmasta-2α,3α,22,23-tetraol], 6α-hydroxy-28-homocastasterone [(22R,23R)-stigmasta-2α,3α,6α,22,23-pentaol], 6β-hydroxy-28-homocastasterone [(22R,23R)-stigmasta-2α,3α,6β,22,23-pentaol], and [(22R,23R)-6α-fluorostigmasta-2α,3α,22,23-tetraol].Results indicate that replacement of the 6-keto moiety by an β or α hydroxyl group led to a decrease in activity, whereas the 6-deoxo analogue showed a very low activity, confirming the importance of an electronegative moiety at C-6 to observe hormonal potency. The 6α-fluorinated analogue elicited a low activity, similar to that of the 6-deoxo analogue.
Keywords: Oryza sativa; Gramineae; Brassinosteroids; Synthesis of fluorinated analogues; Bioactivity;

Further constituents from the bark of Tabebuia impetiginosa by Tsutomu Warashina; Yoshimi Nagatani; Tadataka Noro (589-597).
The polar fraction of the MeOH extract of the bark of Tabebuia impetiginosa afforded twelve compounds, consisting of four iridoid glycosides, one phenylethanoid glycoside, five phenolic gylcosides, and one lignan glycoside. The structures of these compounds were determined using both spectroscopic and chemical methods.Further study on the constituents from the bark of Tabebuia impetiginosa (Mart. ex DC) Standley afforded twelve compounds, consisting of four iridoid glycosides, one phenylethanoid glycoside, five phenolic glycosides, and one lignan glycoside, along with seven known compounds. The structures of these compounds were determined based on the interpretation of their NMR and MS measurements and by chemical evidence.
Keywords: Tabebuia impetiginosa (Mart. ex DC) Standley; Bignoniaceae; Phenylethanoids; Phenolic glycosides; Lignans; Iridoids;

From the liverwort Plagiochila asplenioides, seven sesquiterpenes were isolated and their structures determined by NMR, mass spectrometry and chemical correlations in conjunction with enantioselective gas chromatographyThe essential oil of the liverwort Plagiochila asplenioides from two different locations in Northern Germany were investigated by chromatographic and spectroscopic methods. Seven compounds were isolated by preparative gas chromatography (GC) and their structures investigated by mass spectrometry (MS), NMR techniques and chemical correlations in combination with enantioselective GC. In addition to known constituents, aromadendra-1(10),3-diene, two aromatic sesquiterpene hydrocarbons, bisabola-1,3,5,7(14)-tetraene and bisabola-1,3,5,7-tetraene, three sesquiterpene ethers, muurolan-4,7-peroxide, plagiochilines W and X, in addition to ent-4-epi-maaliol, could be identified as natural compounds for the first time.
Keywords: Plagiochila asplenioides; Liverworts; Sesquiterpene hydrocarbons; Oxygenated sesquiterpenes; abeo-Amorphane/muurolane; seco-Aromadendrane-type sesquiterpenoids;

The formation of glushinskite in the Cactaceae species Opuntia ellisiana constitutes the first evidence of the presence of this biomineral in a plant.The X-ray diffractometric and infrared spectroscopic investigation of crystalline material isolated from the Cactaceae species Opuntia ellisiana shows the presence of a very complex mineral composition, including whewellite (monohydrated calcium oxalate), opal (SiO2), calcite (CaCO3) and glushinskite (dihydrated magnesium oxalate). This is the first report of the presence of magnesium oxalate in plants.
Keywords: Opuntia ellisiana; Cactaceae; Biominerals; Glushinskite; X-ray diffractometry; IR spectra;