Phytochemistry (v.60, #2)

Purification and properties of a lectin from ascomycete mushroom, Ciborinia camelliae by Yumi Otta; Koh Amano; Kano Nishiyama; Akikazu Ando; Shigeru Ogawa; Yoshiho Nagata (103-107).
A lectin was isolated from an ascomycete mushroom, Ciborinia camelliae which was specific to N-acetyl-d-galactosamine. On SDS-polyacrylamide gel electrophoresis; this lectin gave a single band of ∼17-kDa in the presence of 2-mercaptoethanol, but formed dimers, trimers and tetramers in its absence. Amino acid analysis revealed the lectin contained two cysteines and no methionine. The N-terminal sequence was determined up to residue 21, and no homologous proteins including other ascomycete lectins were found.Isolation of a GalNAc-specific lectin, having a molecular mass of 17-kDa from ascomycete Ciborinia camelliae is reported.
Keywords: Ascomycete mushroom; Ascomycete lectin; Ciborinia camelliae lectin;

Gene expression of 5-epi-aristolochene synthase and formation of capsidiol in roots of Nicotiana attenuata and N. sylvestris by Jörg Bohlmann; Einar J. Stauber; Bernd Krock; Neil J. Oldham; Jonathan Gershenzon; Ian T. Baldwin (109-116).
Three new copies of a sesquiterpenoid synthase gene encoding 5-epi-aristolochene synthase (EAS) were cloned as cDNAs from Nicotiana attenuata and functionally characterized after expression of the recombinant enzymes in E. coli. Differential patterns of EAS gene expression and formation of the antimicrobial sesquiterpenoid capsidiol were found in roots and shoots of two species of Nicotiana. EAS is expressed constitutively in roots of N. attenuata and N. sylvestris corresponding with constitutive capsidiol formation in roots. Constitutive expression of EAS and capsidiol accumulation were not detectable in shoots of rosette plants of N. attenuata, but accumulation of terpene synthase transcripts could be induced in shoots by feeding of the tobacco hornworm, Manduca sexta. Constitutive expression of EAS has not been previously reported from N. tabacum, where capsidiol is known as an elicitor- and pathogen-inducible phytoalexin. It is conceivable that capsidiol contributes not only to an inducible defense against pathogens, but also to a constitutive defense in an organ-specific manner in some species of Nicotiana.Expression of epi-aristolochene synthase is constitutive in roots of two species of Nicotiana, associated with constitutive formation of capsidiol. Sesquiterpene synthase expression can be induced by herbivore attack of the tobacco hornworm.
Keywords: Nicotiana attenuata; Nicotiana sylvestris; Solanaceae; Gene expression; Terpene cyclase; 5-epi-Aristolochene; Capsidiol;

Cytokinins and gibberellins in sap exudate of the oil palm by Rachael P Huntley; Laurence H Jones; David E Hanke (117-127).
Sap exudate was obtained from oil palm inflorescences for analysis of cytokinin and gibberellin contentExudates were collected from stumps of pre-anthesis inflorescences of oil palm and analysed for cytokinin and gibberellin content using combined HPLC-ELISA techniques. Three antisera, for zeatin-type, dihydrozeatin-type and isopentenyladenine-type cytokinins, were used in ELISAs to identify members of these three groups of cytokinins. Ribotides, 9-glucosides, free bases and ribosides were detected for each of the groups with zeatin riboside the most abundant cytokinin identified in the exudate. Isopentenyladenine-type and dihydrozeatin-type cytokinins were also identified but at lower levels. In addition, two monoclonal antibodies were used in the development of novel ELISAs for members of the 13-hydroxylated and non-13-hydroxylated families of gibberellins. The new ELISAs allow the determination of gibberellins in smaller amounts of tissue than are required for GC–MS. The most abundant gibberellins identified in exudates were GA19 and GA44, as well as other members of the early 13-hydroxylation pathway. Gibberellins were confirmed by GC–MS. The presence of these types of growth regulators in exudate supplying immature inflorescences suggest they have a role in growth and development of these structures.
Keywords: Elaeis guineensis Jacq.; Palmae; Oil palm; Cytokinins; Gibberellins;

Theacrine (1,3,7,9-tetramethyluric acid) synthesis in leaves of a Chinese tea, kucha (Camellia assamica var. kucha) by Xin-Qiang Zheng; Chuang-Xing Ye; Misako Kato; Alan Crozier; Hiroshi Ashihara (129-134).
Theacrine (1,3,7,9-tetramethyluric acid) and caffeine were the major purine alkaloids in the leaves of an unusual Chinese tea known as kucha (Camellia assamica var. kucha). Endogenous levels of theacrine and caffeine in expanding buds and young leaves were ca. 2.8 and 0.6–2.7% of the dry wt, respectively, but the concentrations were lower in the mature leaves. Radioactivity from S-adenosyl-l-[methyl-14C]methionine was incorporated into theacrine as well as theobromine and caffeine by leaf disks of kucha, indicating that S-adenosyl-l-methionine acts as the methyl donor not only for caffeine biosynthesis but also for theacrine production. [8-14C]Caffeine was converted to theacrine by kucha leaves with highest incorporation occurring in expanding buds. When [8-14C]adenosine, the most effective purine precursor for caffeine biosynthesis in tea (Camellia sinensis), was incubated with young kucha leaves for 24 h, up to 1% of total radioactivity was recovered in theacrine. However, pulse-chase experiments with [8-14C]adenosine demonstrated much more extensive incorporation of label into caffeine than theacrine, possibly because of dilution of [14C]caffeine produced by the large endogenous caffeine pool. These results indicate that in kucha leaves theacrine is synthesized from caffeine in what is probably a three-step pathway with 1,3,7-methyluric acid acting an intermediate. This is a first demonstration that theacrine is synthesized from adenosine via caffeine.Kucha tea contains an unusual purine alkaloid, theacrine. The biosynthesis of theacrine was investigated in young buds and leaves of kucha using radiolabeled S-adenosyl-l-methionine, adenosine and caffeine as substrates.
Keywords: Camellia assamica; Theaceae; Kucha; Chinese tea; Biosynthesis; Caffeine; Purine alkaloids; 1,3,7,9-Tetramethyluric acid; Theacrine;

The freshwater green microalga Parietochloris incisa is the richest known plant source of the polyunsaturated fatty acid (PUFA), arachidonic acid (20:4ω6, AA). While many microalgae accumulate triacylglycerols (TAG) in the stationary phase or under certain stress conditions, these TAG are generally made of saturated and monounsaturated fatty acids. In contrast, most cellular AA of P. incisa resides in TAG. Using various inhibitors, we have attempted to find out if the induction of the biosynthesis of AA and the accumulation of TAG are codependent. Salicylhydroxamic acid (SHAM) affected a growth reduction that was accompanied with an increase in the content of TAG from 3.0 to 6.2% of dry weight. The proportion of 18:1 increased sharply in all lipids while that of 18:2 and its down stream products, 18:3ω6, 20:3ω6 and AA, decreased, indicating an inhibition of the Δ12 desaturation of 18:1. Treatment with the herbicide SAN 9785 significantly reduced the proportion of TAG. However, the proportion of AA in TAG, as well as in the polar lipids, increased. These findings indicate that while there is a preference for AA as a building block of TAG, the latter can be produced using other fatty acids, when the production of AA is inhibited. On the other hand, inhibiting TAG construction did not affect the production of AA. In order to elucidate the possible role of AA in TAG we have labeled exponential cultures of P. incisa kept at 25 °C with [1-14C]arachidonic acid and cultivated the cultures for another 12 h at 25, 12 or 4 °C. At the lower temperatures, labeled AA was transferred from TAG to polar lipids, indicating that TAG of P. incisa may have a role as a depot of AA that can be incorporated into the membranes, enabling the organism to quickly respond to low temperature-induced stress.Triacylglycerols may have a role as a depot of arachidonic acid in the alga Parietochloris incisa.
Keywords: Parietochloris incisa; Trebuxiophyceae; Arachidonic acid; LC-PUFA; Salicylhydroxamic acid; SAN 9785; Triacylglycerols;

Integration of ethnobotany and phytochemistry by Otto R. Gottlieb; Maria Renata de M.B. Borin; Nei Regis S. de Brito (145-152).
The major challenge of this paper comprises an attempt to compare the wealth of folk-information based on “traditional knowledge” with the efficacy of a chemo-biological background based on scientific knowledge. Surprisingly, correlations between plant uses (as food and medicine) and evolutionary status according to morphology (as assigned by Sporne indices) and metabolism (as assigned by diversity of phytochemicals) obey strikingly uniform systematic and evolutionary trends. The resulting patterns suggest a dynamic chemical mechanism for the bioactivity in plants regulated by the antagonistic gallate/caffeate pair. This finding is an important step toward the construction of a coherent chemo-biological language by a dynamic holistic quantitative methodology, one of the most potent prospects for understanding the functioning of nature.The translation of 12 American and African ethnobotanical inventories of angiosperms by an evolutionary language allows integration with phytochemical databases, leading to new insights into the mechanisms of bioactivity.
Keywords: Ethnobotany; Angiosperms; Medicinal species; Edible species; Antagonisms; Gallic acid; Caffeic acid;

Accumulation and detoxification of lead ions in legumes by Aneta Piechalak; Barbara Tomaszewska; Danuta Baralkiewicz; Arleta Malecka (153-162).
This study focuses on lead accumulation in roots, stems and leaves of three plant species of the Fabacea family: Vicia faba, Pisum sativum and Phaseolus vulgaris grown hydroponically in a medium supplemented with 1 mM concentration of lead. The largest amount of lead, up to 75 mg Pb/g dry weight, was accumulated in roots of P. vulgaris. The highest rate of Pb ions uptake from the medium took place during the first 10 h of incubation with lead and after 96 h of incubation lead content in the medium decreased by half. Thus, it was suggested that P. vulgaris could be used in rhizofiltration—the use of plant roots to absorb pollutants from water contaminated with lead. At the same time we studied the influence of lead on acid soluble thiol, glutathione, homoglutathione contents and the synthesis of phyto- and homophytochelatins in roots of V. faba, P. sativum and P. vulgaris grown hydroponically. Activation of the detoxicative-phytochelatin system was observed in the cytosol of root cells of the tested plants. This system was composed of phytochelatins (PCs) in roots of V. faba, homophytochelatins (hPCs) in P. vulgaris roots and both PCs and hPCs in P. sativum roots. The total content of PCs and hPCs in roots of P. sativum was very high and reached around 4800 (expressed in nmol SH×g−1FW) and induction of their synthesis occurred after only 2 h of treatment with 1 mM Pb.This study focuses on lead accumulation of three plant species of the Fabacea family grown hydroponically in a medium supplemented with 1 mM concentration of lead.
Keywords: Phytoremediation; Phytochelatins; Homophytochelatins; Detoxification; Lead ions;

Antifeedant activity of an anthraquinone aldehyde in Galium aparine L. against Spodoptera litura F. by Masanori Morimoto; Kumiko Tanimoto; Akiko Sakatani; Koichiro Komai (163-166).
The insect antifeedant anthraquinone aldehyde nordamnacanthal (1,3-dihydroxy-anthraquinone-2-al) was identified in Galium aparine L., and isolated from the root powder of akane (Rubia akane), a member of the Rubiaceae. Structure–activity relationship (SAR) studies using a series of anthraquinone analogues suggested that the aldehyde group on the anthraquinone was more important than the quinone moiety for antifeedant activity against the common cutworm (Spodoptera litura). High levels of nordamnacanthal were found in the seed leaf stage and in callus tissue induced from seedlings of G. aparine, but its concentration decreased with plant development. Since these compounds are natural pigments for dying textiles, we also evaluated the antifeedant activity against the carpet beetle (Attagenus japonicus ), a textile pest was also evaluated. While nordamnacanthal had strong antifeedant activity against the common cutworm, it did not show any antifeedant activity against the carpet beetle. The most effective antifeedant against the carpet beetle was the major constituent in the extract of R. trictorum, lucidin-3-O-primeveroside, a food pigment.The insect antifeedant anthraquinone aldehyde nordamnacanthal (1,3-dihydroxy-anthraquinone-2-al) was identified in Galium aparine L. A structure–activity relationship (SAR) study using anthraquinone analogues suggested that the aldehyde group on the anthraquinone was more important than the quinone moiety for antifeedant activity against the common cutworm (Spodoptera litura).
Keywords: Galium aparine L; Rubia akane; Rubiaceae; Anthraquinones; Nordamnacanthal; Spodoptera litura; Attagenus japonicus;

Antiplasmodial activity of sesquilignans and sesquineolignans from Bonamia spectabilis by Carola Kraft; Kristina Jenett-Siems; Inga Köhler; Britta Tofern-Reblin; Karsten Siems; Ulrich Bienzle; Eckart Eich (167-173).
Phytochemical re-investigation of the aerial parts of Bonamia spectabilis (Convolvulaceae) led to the isolation of four minor tetrahydrofuran-type sesquilignans (bonaspectins E–H) together with the known neolignan virolongin A and the known lignan rel-(7S,8R,7′R,8′R)-3,3′,4,4′,5,5′-hexamethoxylignan. Their structures were established on the basis of spectral data. These six compounds as well as further seven lignanoids from B. spectabilis, characterised previously, were tested for their antiplasmodial activity against a chloroquine-sensitive strain (PoW) and a chloroquine-resistant clone (Dd2) of Plasmodium falciparum. Bonaspectin C 4″-O-glucoside, its aglycone, and bonaspectin D 4″-O-glucoside revealed the highest antiplasmodial activities (IC50 values: 1.3, 2.0, 6.5 μM [PoW]; 1.7, 4.6, 3.7 μM [Dd2], respectively).Four minor tetrahydrofuran-type sesquilignans (bonaspectins E–H) together with virolongin A and rel-(7S,8R,7′R,8′R)-3,3′,4,4′,5,5′-hexamethoxylignan have been isolated from the aerial parts of Bonamia spectabilis (Convolvulaceae). These compounds as well as further lignanoids of this plant species were tested for their antiplasmodial activity in vitro.
Keywords: Bonamia spectabilis; Convolvulaceae; Lignans; Sesquilignans; Sesquineolignans; Bonaspectin C 4″-O-glucoside; Antiplasmodial activity; Plasmodium falciparum;

Spermine alkaloids from Albizia adinocephala with activity against Plasmodium falciparum plasmepsin II by Simon P.B Ovenden; Shugeng Cao; Chungyan Leong; Horst Flotow; Mahabir P Gupta; Antony D Buss; Mark S Butler (175-177).
Crude MeOH extracts from the stem bark and leaves of a Panamanian specimen of Albizia adinocephala (Leguminosae) were found to inhibit the malarial enzyme plasmepsin II. Bioassay guided fractionation led to the isolation of two new bioactive spermine alkaloids, budmunchiamines L4 and L5.Bioassay guided fractionation of a MeOH extract from the stem bark and leaves of Albizia adinocephala led to the isolation of two bioactive spermine alkaloids, budmunchiamines L4 and L5.
Keywords: Albizia adinocephala; Leguminosae; Stem bark; Leaves; Spermine alkaloid; Budmunchiamine; Plasmepsin II; Malaria;

Isopestacin, an isobenzofuranone from Pestalotiopsis microspora, possessing antifungal and antioxidant activities by Gary Strobel; Eugene Ford; Jeerepun Worapong; James K. Harper; Atta M. Arif; David M. Grant; Peter C.W. Fung; Raymond Ming Wah Chau (179-183).
Isopestacin is an isobenzofuranone obtained from the endophytic fungus Pestalotiopsis microspora. While a few other isobenzofuranones are known from natural sources, isopestacin is the only one having a substituted benzene ring attached at the C-3 position of the furanone ring. The compound was isolated from culture broths of the fungus and crystallized and its structure was determined by X-ray crystallography. Both proton and carbon NMR spectral assignments are also reported for isopestacin. This compound possesses antifungal activity and, as measured by electron spin resonance specroscopy, it also behaves as an antioxidant scavenging both superoxide and hydroxy free radicals.An isobenzofuranone from a culture of Pestalotiopsis microspora possessing antifungal and antioxidant activities is described.
Keywords: Isobenzofuranone; Antioxidant; Antimycotic; Endophyte; Fungus; X-ray; NMR;

Triterpenoid saponins from Oreopanax guatemalensis by F.R. Melek; Toshio Miyase; S.M. Abdel-Khalik; M.H. Hetta; I.I. Mahmoud (185-195).
Seven oleanane-type saponins were isolated from the leaves and stems of Oreopanax guatemalensis, together with ten known saponins of lupane and oleanane types. The new saponins were respectively characterized as 3-O-α-l-arabinopyranosyl echinocystic acid 28-O-[α- l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl] ester, 3-O-β-d-glucopyranosyl 3β-hydroxy olean-11,13(18)-dien-28-oic acid 28-O-[α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl-(1→6)-β- d-glucopyranosyl]ester, 3-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]3β-hydroxy olean-11,13(18)-dien-28-oic acid 28-O-[α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl] ester, 3-O-[α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl]3β, 23 dihydroxy olean-18-en-28-oic acid 28-O-[α-l-rhamnopyranosyl-(1→4)-β-d-6-O-acetyl glucopyranosyl-(1→6)-β-d-glucopyranosyl]ester, 3-O-[α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl] hederagenin 28-O-{α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl-(1→6)-[β-d-xylopyranosyl-(1→2 )-]β-d-glucopyranosyl}ester, 3-O-[α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl]hederagenin 28-O-{α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl-(1→6)-[β-d-glucopyranosyl-(1→2)-]β-d-glucopyranosyl} ester and 3-O-[α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl] hederagenin 28-O-{α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl-(1→6)-[α-l-arabinofuranosyl-(1→2)]-β-d-glucopyranosyl} ester. The structures were determined by spectral analyses. The NMR assignments were made by means of HOHAHA, 1H–1H COSY, HMQC, HMBC spectra and NOE difference studies.Seven oleanane-type saponins were isolated and identified from the leaves and stems of Oreopanax guatemalensis. The structures of the compounds were determined by spectral analysis.
Keywords: Oreopanax guatemalensis; Araliaceae; Triterpenoid saponin;

Three labdane diterpenoids from Aframomum sceptrum (Zingiberaceae) by Christabel Tomla; Pierre Kamnaing; Godfred A Ayimele; Eric A Tanifum; Apollinaire Tsopmo; Pierre Tane; Johnson F Ayafor; Joseph D Connolly (197-200).
Three labdane diterpenoids, 8β,17-epoxy-3β,7β-dihydroxy-12(E)-labden-16,15-olide (1), methyl 8β,17-epoxy-3β,7β,15-trihydroxy-12(E)-labden-16-oate (2) and 3β,7β,8β,12ζ,17-pentahydroxylabdan-16,15-olide (3) have been isolated from the seeds of Afromomum sceptrum K. Schum (Zingiberaceae) and their structures assigned on the basis of their spectroscopic properties. Nerolidol, and the known flavonoids 3-acetoxy-4′,5,7-trihydroxyflavanone, and 3,4′,5,7-tetrahydroxyflavanone were also obtained.8β,17-Epoxy-3β,7β-dihydroxy-12(E)-labden-16,15-olide and two closely related labdane diterpenoids have been isolated from Aframomum sceptrum.
Keywords: Aframomum sceptrum; Zingiberaceae; Labdane diterpenoids;