Phytochemistry (v.59, #3)

Transport of 1-kestose across the tonoplast of Jerusalem artichoke tubers by Horacio G Pontis; Pedro González; Ed Etxeberria (241-247).
The capacity for 1-kestose uptake into the vacuole of fructan storing Jerusalem artichoke tubers was investigated. 1-kestose serves both as building block for fructan initiation and as a fructose donor for chain elongation. Tonoplast vesicles were isolated from actively storing tubers, and their vesicles were capable of transporting sucrose in a manner indicative of a sucrose/H+ antiport. Under similar conditions, 1-kestose was not taken up by vesicles energized by either a pH jump or in the presence of ATP. When added together at 2 mM, sucrose uptake was not affected by the presence of 1-kestose. The data argues against the possible synthesis of 1-kestose in the cytosol and subsequent transport to the vacuole. The data also presents definite evidence for the existence a mechanism for sucrose accumulation in fructan storing vacuoles.The properties of tonoplast transport in developing Jerusalem artichoke tubers as related to fructan initiation were investigated. Tonoplast enriched vesicles were able to accumulate sucrose by an apparent sucrose/H+ antiport. 1-Kestose was not transported across the tonoplast either by a kestose/H+ antiport or directly energized by ATP. Sucrose transport was not affected by the presence of 1-kestose.
Keywords: Fructan synthesis; Tonoplast transport; Fructan initiation; Vacuole;

Xylan and callose was immunolocated to secondary thickenings in differentiating xylem cells. A xylan synthase-associated polypeptide was immunolocated throughout Golgi stacks in these cells. A callose synthase-associated polypeptide was immunolocated to the surface of the growing thickenings and the plasmalemma.The Golgi apparatus of plant cells is thought to be the main site of synthesis of cell wall matrix polysaccharides and the terminal glycosylation of glycoproteins. Much of this evidence still depends on earlier biochemical studies employing subcellular fractionation. However acquiring pure Golgi membranes is still difficult and the question of spatial organisation of glycosyl transferases can be addressed by immunolocation of the enzymes. An antibody to a xylan synthase-associated polypeptide from French bean, the enzyme which synthesises the core polysaccharide for secondary wall xylan, has been raised and shown to inhibit its activity. Xylan is deposited in secondary thickenings and the xylan synthase was only detected in appreciable amounts in developing xylem cells. The location within the Golgi stack was observed throughout the dictyosomes. Some enzyme subunits were also detected in post-Golgi vesicles. A second antibody to a non-catalytic M r 65 000 subunit of β 1,3- glucan (callose) synthase was used for a comparative study. Although the bulk of this enzyme has been detected in previous studies at plasmamembrane-wall interfaces in sieve plates and stressed tissue, a Golgi-location can be observed in root tip meristematic cells during cell plate formation. The enzyme was present throughout the stacks. Callose was also immunolocated in a similar manner to xylan in secondary walls and thickenings and in pits in developing xylem. In these cells, the callose synthase was detected at the surface of the growing thickenings and the plasmamembrane within the pits.
Keywords: Phaseolus vulgaris; Leguminosae; Xylogenesis; Cell wall polysaccharides; Xylan synthase; Callose synthase; Immunolocation;

Four cDNA clones encoding two large subunits and two small subunits of the starch regulatory enzyme ADP-glucose pyrophosphorylase (AGPase) were isolated from a chickpea (Cicer arietinum L.) stem cDNA library. DNA sequence and Southern blot analyses of these clones, designated CagpL1, CagpL2 (large subunits) and CagpS1 and CagpS2 (small subunits), revealed that these isoforms represented different AGPase large and small subunits. RNA expression analysis indicated that CagpL1 was expressed strongly in leaves with reduced expression in the stem. No detectable expression was observed in seeds and roots. CagpL2 was expressed moderately in seeds followed by weak expression in leaves, stems and roots. Similar analysis showed that CagpS1 and CagpS2 displayed a spatial expression pattern similar to that observed for CagpL2 with the exception that CagpS1 showed a much higher expression in seeds than CagpS2. The spatial expression patterns of these different AGPase subunit sequences indicate that different AGPase isoforms are used to control starch biosynthesis in different organs during chickpea development.ADP-glucose pyrophosphorylase is the key regulatory enzyme in starch biosynthesis. The presence of multiple gene sequences, which display distinct spatial expression patterns during growth and development, indicates that different AGPase isoforms are used to control starch biosynthesis in different organs during chickpea development.
Keywords: ADP-glucose pyrophosphorylase; Chickpea (Cicer arietinum L.);

The incorporation of 13C labeled 1-deoxy-d-xylulose into the monoterpene bornyl acetate, the sesquiterpene cubebanol, and the diterpene phytol has been studied in axenic cultures of the liverwort Conocephalum conicum. Quantitative 13C NMR spectroscopic analysis of the labeling patterns of the sesquiterpene indicated a possible degradation of 1-deoxy-d-xylulose to acetate and subsequent incorporation via the mevalonic acid pathway. In bornyl acetate, the labeling occurred only in the acetate moiety whereas the isoprene units remained unlabelled. The isoprene units of the diterpene phytol showed incorporation of intact deoxy-d-xylulose. These results indicate the involvement of both IPP biosynthetic pathways and two independently operating compartments/cell types with MEP pathway machinery. One MEP compartment is presumably the plastid where phytol is formed; the second, involved in the build-up of the isoprene part of bornyl acetate, might be located in the oil cells. The acetylation of borneol to bornyl acetate in turn occurs in a cellular compartment that is not involved in the build-up of the isoprene units of borneol.[1-13C]1-Deoxy-d-xylulose has been incorporated into the monoterpene bornyl acetate, the sesquiterpene cubebanol, and the diterpene phytol in the liverwort Conocephalum conicum. It could be demonstrated that bornyl acetate and phytol are formed in different MEP pathway containing cellular compartments.
Keywords: Liverwort; Conocephalum conicum; Hepaticae; Isopentenyl diphosphate; Mevalonic acid pathway; Methylerythritol phosphate pathway; Bornyl acetate; Cubebanol; Phytol; [1-13C]1-Deoxy-d-xylulose;

Flavonoid 5-glucosides from the cocoon shell of the silkworm, Bombyx mori by Yasumori Tamura; Ken-ichi Nakajima; Ken-ichi Nagayasu; Chiyuki Takabayashi (275-278).
The flavonoid 5-glucosides, quercetin 5,4′-di-O-β-d-glucopyranoside and quercetin 5,7,4′-tri-O-β-d-glucopyranoside, together with the known quercetin 5-O-β-d-glucopyranoside, were isolated from the cocoon shell of the silkworm, Bombyx mori. The structures were identified by spectroscopic analysis. These flavonoid glucosides were not present in mulberry leaves, the silkworm's only food, and they are considered to be metabolites produced by the silkworm.The flavonoid 5-glucosides, quercetin 5,4′-di-O-β-d-glucopyranoside (1) and quercetin 5,7, 4′-tri-O-β-d-glucopyranoside (2) together with the known quercetin 5-O-β-d-glucopyranoside were isolated from the cocoon shell of the silkworm, Bombyx mori.
Keywords: Bombyx mori; Silkworm; Cocoon; Flavonoid; Quercetin 5,4′-di-O-β-d-glucopyranoside; Quercetin 5,7,4′-tri-O-β-d-glucopyranoside; Quercetin 5-O-β-d-glucopyranoside;

Serine proteinase inhibitors in the Compositae: distribution, polymorphism and properties by Alexander V. Konarev; Irina N. Anisimova; V.A. Gavrilova; T.E. Vachrusheva; G.Yu. Konechnaya; Mervyn Lewis; Peter R. Shewry (279-291).
Multiple molecular forms of inhibitors of trypsin (TI) and chymotrypsin (CI), which are typical digestive enzymes of insects, mammals and micro-organisms, and subtilisin (SI), a proteinase of many bacteria and phytopathogenic fungi, were identified in seeds and vegetative organs of the majority of 128 wild and cultivated species representing 65 genera of three of the subfamilies of the Compositae. Inhibitors with M r ranging from 7450 to 7800 and combining activities towards subtilisin and trypsin and/or chymotrypsin (T/C/SI) had the widest distribution and may be involved in plant defense mechanisms. They were found in many species of the subfamilies Carduoideae (genera Carthamus, Centaurea, Cirsium), Cichorioideae (Lactuca, Taraxacum) and Asteroideae (Helianthus, Cosmos, Bidens). Partial amino acid sequencing showed that the safflower (Carthamus tinctorius) T/C/SI and Cosmos bipinnatus T/C/SI, T/SI and C/SI belonged to the potato I inhibitor family. The most active, variable and heterogeneous inhibitors were found in species of the tribe Heliantheae, which is placed in the evolutionary advanced subfamily Asteroideae. Seeds of Helianthus species, Eclipta prostrata, Gailardia aristata, Zinnia elegans and Silphium perfoliatum contained various TI with M r ranging from 1500 to 14,750, with some also containing SI. H. annuus seeds contain a unique cyclic TI of M r 1514 and similar TI were also present in other Helianthus spp. and the related species Tithonia diversifolia. Zinnia elegans contained a TI with M r 11,350 which appeared to represent a novel type of inhibitor distantly related to the cereal subgroup of Bowman–Birk inhibitors. TI and T/SI varied widely in H. annuus lines and wild Helianthus species in their presence or absence and composition. Similar T/SI components were found in the cultivated diploid H. annuus and annual diploid species with the B genome but not in perennials with the A genome. Some T/SI, SI and TI were detected in vegetative organs of sunflower and other Compositae. Studies of the polymorphism and distribution of proteinase inhibitors are relevant to the evolution of protective protein systems and the mechanisms of resistance to pathogenic organisms in the Compositae and other plants.Seeds of 128 species of the Compositae contain proteinase inhibitors ranging in M r from 1500 to 15,000 with activity against trypsin, chymotrypsin and subtilisin, including novel forms restricted to specific taxonomic groups.
Keywords: Compositae; Proteinase inhibitors; Polymorphism;

Chemosystematic investigations of irregular diterpenes in Anisotome and related New Zealand Apiaceae by Christian Zidorn; Sonja Sturm; John W. Dawson; John W. van Klink; Hermann Stuppner; Nigel B. Perry (293-304).
A chemosystematic HPLC–UV and HPLC–MS investigation of New Zealand members of the Apiaceae was performed. Diterpenes were identified and quantified in methanolic extracts from subaerial parts of 28 taxa and 54 samples of Aciphylla, Anisotome, Apium, Gingidia, Lignocarpa, Oreomyrrhis, and Scandia. Six diterpenes (12, 47) and four polyacetylenes (811) were identified. The known compounds were the diterpenes anisotomenoic acid 1, anisotomene-1-ol 2, 16-acetoxyanisotomenoic acid 4 and anisotomene-1,12-diol 5; and the polyacetylenes falcarinol 8, falcarindiol 9, (+)-9(Z),17-octadecadiene-12,14-diyne-1,11,16-triol 10, and (+)-9(Z),17-octadecadiene-12,14-diyne-1,11,16-triol 1-acetate 11. New irregular diterpenes 13,14-dihydroanisotom-12E-ene-1,14-diol 6 and 14-methoxy-13,14-dihydroanisotom-12E-ene-1-ol 7 were isolated from A. haastii. Isomers of the new semi-synthetic diterpene 16-hydroxyanisotomenoic acid 3 were detected in extracts of Anisitone flexuosa. Structure elucidation was performed by HR mass spectrometry and 1D and 2D NMR spectroscopy. In crude extracts, compounds were identified by their HPLC retention times and their on-line HPLC–UV and MS spectra. Anisotomene diterpenes occurred in eight out of 16 species of the genus Anisotome, but were not detected in any of the other genera. In contrast, polyacetylenes were present in all the genera investigated.Anisotomene type diterpenes were used as chemosystematic markers in Anisotome and other New Zealand genera of the Apiaceae. In the course of the investigations three derivatives were isolated from A. flexuosa and A. haastii. In crude extracts compounds were identified by HPLC-MS and quantified by HPLC-UV.
Keywords: Anisotome; Aciphylla; Gingidia; Lignocarpa; Scandia; Apiaceae; Chemosystematics; Diterpenes; Polyacetylenes;

Essential fatty acids and phenolic acids from extracts and leachates of southern cattail (Typha domingensis P.) by Maria T Gallardo-Williams; Cherie L Geiger; Joseph A Pidala; Dean F Martin (305-308).
We have been able to isolate several phytotoxic compounds from aqueous extracts and leachates of cattails (Typha domingensis) using activated charcoal as an absorbant, followed by successive extraction with organic solvents, analysis by GC/MS, and structural elucidation by NMR spectroscopy when possible. The phytotoxins were identified as essential fatty acids (linoleic acid and α-linolenic acid) and phenolic compounds of known phytotoxic activity (caffeic acid from the aqueous extracts; caffeic, p-coumaric, and gallic acid from the leachates). Both extracts and the phytotoxins in the extracts have the potential of inhibiting the growth and chlorophyll production of several ecologically relevant species.Phytotoxic essential fatty acids and phenolic compounds have been isolated from cattail aqueous extracts and leachates.
Keywords: Typha domingensis; Typhaceae; Cattail; Phytotoxins; Fatty acids; Phenolics;

Phytotoxicity and mammalian cytotoxicity of macrocyclic trichothecene mycotoxins from Myrothecium verrucaria by H.K Abbas; B.B Johnson; W.T Shier; H Tak; B.B Jarvis; C.D Boyette (309-313).
Macrocyclic trichothecene toxins produced by Myrothecium verrucaria (a phytopathogen of interest in biological weed control) and the non-trichothecene toxin atranone B from Stachybotiys atra were tested for phytotoxicity in duckweed (Lemna pausicostata L.) plantlet cultures and kudzu (Pueraria lobata L.) leaf disc assays, and for mammalian cytotoxicity in four cultured cell lines. Roridin E and H, epi-isororidin E, and verrucarin A and J were phytotoxic (half-maximal effect in the concentration range 0.1–9.7 μM on duckweed and 1.5–>80 μM on kudzu) and cytotoxic to mammalian cell lines (half-maximal inhibition of proliferation in the concentration range 1–35 nM). Trichoverrins A and B and atranone B were moderately phytotoxic (half-maximal effect in the concentration range 1 9–69 μM on duckweed and 13–>80 μM on kudzu) and weakly cytotoxic with mammalian cell lines (half-maximal inhibition of proliferation in the concentration range 0.3–>2 μM).Macrocyclic trichothecenes exhibit both phytotoxicity and mammalian cytotoxicity.
Keywords: Kudzu, Pueraria montana Leguminosae, Macrocyclic trichothecenes; Mycotoxins; Natural products; Mycoherbicide; Mvrothecium verrucaria; Phytotoxins; Phytotoxicity; Cytotoxicity; Biological control;

Production of bioactive triterpenes by Eriobotrya japonica calli by Shoko Taniguchi; Yoko Imayoshi; Eri Kobayashi; Yoshie Takamatsu; Hideyuki Ito; Tsutomu Hatano; Hiroshi Sakagami; Harukuni Tokuda; Hoyoku Nishino; Daigo Sugita; Susumu Shimura; Takashi Yoshida (315-323).
Callus tissue cultures induced from an axenic leaf of Eriobotrya japonica (Rosaceae) produced triterpenes in large amounts (ca. 50 mg/g dry wt). Nine triterpenes were characterized as ursolic acid, oleanolic acid, 2α-hydoxyursolic acid, maslinic acid, tormentic acid, 2α, 19α-dihydroxy-3-oxo-urs-12-en-28-oic acid, hyptadienic acid and a mixture of 3-O-cis-p-coumaroyltormentic acid and 3-O-trans-p-coumaroyltormentic acid. The triterpene composition in the callus tissues was noticeably different from that in intact leaves. The contents of tormentic acid with antidiabetic action, and 2α, 19α-dihydroxy-3-oxo-urs-12-en-28-oic acid with anti-HIV activity, were much larger than those in the intact leaves. All of the triterpenes isolated from the callus tissues showed an inhibitory effect comparable to (−)-epigallocatechin gallate (EGCG) of green tea on the activation of Epstein–Barr virus early antigen (EBV-EA) induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). 2α, 19α-Dihydroxy-3-oxo-urs-12-en-28-oic acid was the most potent inhibitor among them and caused a significant delay of two-stage carcinogenesis on mouse skin.Callus tissue of Eriobotrya japonica (Rosaceae) producing triterpenes in large amounts (ca. 50 mg/g dry wt) was established. All of the triterpenes from the calli showed inhibitory effect on the activation of Epstein–Barr virus early antigen (EBV-EA) induced by 12-O-tetradecanoylphorbol-13-acetate (TPA).
Keywords: Eriobotrya japonica; Rosaceae; Callus tissue culture; Triterpene; Epstein–Barr virus; Two-stage carcinogenesis;

Natural anti-HIV agents—part I: (+)-demethoxyepiexcelsin and verticillatol from Litsea verticillata by Vu Dinh Hoang; Ghee Teng Tan; Hong-Jie Zhang; Pamela A Tamez; Nguyen Van Hung; Nguyen Manh Cuong; D.Doel Soejarto; Harry H.S Fong; John M Pezzuto (325-329).
The eudesmane sesquiterpenoid, verticillatol (1), as well as the lignan, (+)-5′-demethoxyepiexcelsin (2), and a known lignan, (+)-epiexcelsin (3), were isolated from Litsea verticillata Hance. Lignan 2 showed moderate anti-HIV activity with an IC50 value of 16.4 μg/ml (42.7 μM), while the known lignan 3 was inactive up to a concentration of 20 μg/ml (48.3 μM). Compound 1 demonstrated weak activity with an IC50 value of 34.5 μg/ml (144.7 μM) while being devoid of cytotoxicity at 20 μg/ml. The structures were elucidated by 1D and 2D NMR spectroscopy, and the absolute configuration of the new sesquiterpenoid was determined by the generation of Mosher esters.The eudesmane sesquiterpenoid, verticillatol (1), and the lignan, (+)-5′-demethoxyepiexcelsin (2), as well as a known lignan, (+)-epiexcelsin (3), were isolated from Litsea verticillata Hance. 1 and 2 showed anti-HIV activity with IC50 values of 34.5 and 16.4 μg/ml, respectively, while being devoid of cytotoxicity at 20 μg/ml. The structures were elucidated by 1D and 2D NMR spectroscopy, and the absolute configuration of 1 was determined by the generation of Mosher esters.
Keywords: Litsea verticillata; Lauraceae; Verticillatol; Sesquiterpene; (+)-5′-Demethoxyepiexcelsin; Lignan; Sesquiterpene; 2D NMR; Absolute structure; Anti-HIV activity; HOG.R5; Reporter cell line; Green fluorescent protein;

Macrocyclic diterpenes from Euphorbia nivulia by V Ravikanth; V.L Niranjan Reddy; T Prabhakar Rao; P.V Diwan; S Ramakrishna; Y Venkateswarlu (331-335).
The latex of Euphorbia nivulia afforded two ingol diterpenes 3,12-diacetyl-8-benzoylingol (4) and 3,12-diacetyl-7-benzoyl-8-nicotinylingol (5) along with three known ingol diterpenes 1, 2,and 3, and two known triterpenes cycloart-25-en-3β-ol and cyclonivulinol. Their structures have been assigned on the basis of their structural data as well as their acetylated products. The diterpenes 15 were tested for the LPS induced PGE2 inhibition activity.The latex of Euphorbia nivulia afforded two ingol diterpenes 3,12-diacetyl-8-benzoylingol (4) and 3,12-diacetyl-7-benzoyl-8-nicotinylingol (5) along with three known ingol diterpenes 1, 2, and 3, and two known triterpenes cycloart-25-en-3β-ol and cyclonivulinol. Their structures have been assigned on the basis of their structural data as well as their acetylated products. The diterpenes 15 were tested for the LPS induced PGE2 inhibition activity.
Keywords: Euphorbia nivulia; Euphorbiaceae; Ingol diterpenes; 3,12-Diacetyl-8-benzoylingol; 3,12-Diacetyl-7-benzoyl-8-nicotinylingol; In vitro PGE2 assay;

Three isoflav-3-enes and a 2-arylbenzofuran from the root bark of Erythrina burttii by Abiy Yenesew; Jacob O Midiwo; Salome M Guchu; Matthias Heydenreich; Martin G Peter (337-341).
From the root bark of Erythrina burttii three isoflav-3-enes, 7,4′-dihydroxy-2′-methoxy-6-(1″,1″-dimethylallyl)isoflav-3-ene (trivial name, burttinol-A), 4′-hydroxy-2′-methoxy-2″,2″-dimethylpyrano[5″,6″:8,7]isoflav-3-ene (trivial name, burttinol-B), 7,4′-dihydroxy-2′-methoxy-8-(3″,3″-dimethylallyl)isoflav-3-ene (trivial name, burttinol-C), and 2-arylbenzofuran, 6,4′-dihydroxy-2′-methoxy-5-(1″,1″-dimethylallyl)-2-arylbenzofuran (trivial name, burttinol-D) were isolated. In addition, the known compounds, abyssinone V-4′-methyl ether, bidwillol A, calopocarpin, erybraedin A, erythrabyssin II, isobavachalcone, phaseollidin and phaseollin were identified. The structures were determined on the basis of spectroscopic evidence.Graphic
Keywords: Erythrina burttii; Leguminosae; Root bark; Isoflav-3-enes; 2-Arylbenzofuran; Burttinol-A; Burttinol-B; Burttinol-C; Burttinol-D;

The structure of the major anthocyanin in Arabidopsis thaliana by Stephen J Bloor; Sharon Abrahams (343-346).
The major anthocyanin in the leaves and stems of Arabidopsis thaliana has been isolated and shown to be cyanidin 3-O-[2-O(2-O-(sinapoyl)-β-d-xylopyranosyl)-6-O-(4-O-(β-d-glucopyranosyl)-p-coumaroyl-β-d-glucopyranoside] 5-O-[6-O-(malonyl) β-d-glucopyranoside]. This anthocyanin is a glucosylated version of one of the anthocyanins found in the flowers of the closely related Matthiola incana.The major anthocyanin in the leaves and stems of Arabidopsis thaliana has been isolated and shown to be cyanidin 3-O-[2-(2-(sinapoyl)xylosyl)-6-O-(4(glucosyl)-p-coumaroyl)glucoside]5-[6-O-(malonyl)glucoside].
Keywords: Arabidopsis thaliana; Brassicaceae; Anthocyanin; Acylated cyanidin 3-sambubioside-5-glucoside; Malonic acid; Sinapic acid; p-Coumaric acid;

HPLC/PDA/ESI/MS and HPLC/PDA/ESI/MS/MS are used to profile, identify, and compare triterpene saponins isolated from Medicago sativa (alfalfa) and Medicago truncatula roots.Triterpene saponins isolated from Medicago sativa (alfalfa) and Medicago truncatula roots were separated, profiled and identified using an optimized, reversed-phase HPLC with on-line photodiode array detection and electrospray ionization mass spectrometry method (HPLC/PDA/ESI/MS). ESI source polarity and solvent conditions were compared. The effects of these parameters on mass spectral attributes were determined. Ion structures were confirmed using tandem mass spectrometry (MS/MS). Fifteen saponins were identified in alfalfa (cultivars Apollo, Radius, and Kleszczewska) based upon negative-ion HPLC/PDA/ESI/MS, HPLC/PDA/ESI/MS/MS and literature data. In addition, the identification of two new malonated saponins in alfalfa are proposed. Negative-ion HPLC/PDA/ESI/MS and HPLC/PDA/ESI/MS/MS spectra were utilized along with HPLC retention times to profile and identify 27 saponins in M. truncatula (cultivar Jemalong, A17). M. truncatula yielded a much more complex mixture of saponins than observed for alfalfa. The authors are not aware of any previous reports identifying saponin glycosides in M. truncatula.
Keywords: Medicago sativa; Alfalfa; Medicago truncatula; Fabaceae; Legumes; Metabolite profiling; Saponins; Malonyl saponins; High performance liquid chromatography (HPLC); Electrospray ionization (ESI); Mass spectrometry (MS);

Corrigendum to “Bacopaside I and II: two pseudojujubogenin glycosides from Bacopa monniera” [Phytochemistry 58 (2001) 553–556] by Ajit K Chakravarty; Tapas Sarkar; Kazuo Masuda; Kenji Shiojima; Takahisa Nakane; Nobuo Kawahara (365).