Phytochemistry (v.59, #1)

Predicting the substrates of cloned plant O-methyltransferases by Gudrun Schröder; Elke Wehinger; Joachim Schröder (1-8).
Plant O-methyltransferases (OMTs) have important roles in secondary metabolite biosynthesis. Sequencing projects and homology-based cloning strategies yield sequences for proteins with similarities to known OMTs, but the identification of the physiological substrates is not trivial. We investigated with a cDNA cloned from Catharanthus roseus the possibilities for predicting the substrates of OMTs, using the information from previous work and two newly identified motifs that were based on information from the crystal structures of two plant OMTs. The results, confirmed by functional analysis of the recombinant protein, indicated that a careful analysis of the deduced protein sequence can provide clues for predicting the substrates of cloned OMTs.Sequencing projects and homology-based PCR cloning strategies yield sequences for putative O-methyltransferases, but the identification of the physiological substrates is not trivial. We test with a cDNA from Catharanthus roseus whether the previously available information and two newly identified protein motifs are sufficient for reliable predictions of the substrate specificity.
Keywords: Catharanthus roseus; Apocynaceae; Madagascar periwinkle; Homology based cDNA cloning; Recombinant protein expression; O-Methyltransferase; Phenylpropanoid; Flavonoids; Caffeic acid;

Dextrorotatory 1-amino-3′,4′-dichlorobenzylphosphonic acid was found to be a potent inhibitor of the plant enzyme phenylalanine ammonia-lyase both in vitro and in vivo from among the ring-substituted 1-aminobenzylphosphonic acids and other analogues of phenylglycine. A structure activity relationship analysis of the results obtained permits predictions on the geometry of the pocket of the enzyme and is a basis in the strategy of better inhibitor synthesis.A set of ring-substituted 1-aminobenzylphosphonic acids, and a number of substituted phenylglycines, were synthesised and evaluated as inhibitors of phenylalanine ammonia-lyase both in vitro and in vivo.
Keywords: Phenylalanine ammonia-lyase; 1-Aminoalkylphosphonic acids; Enzyme inhibitors;

Flavonoid gene expression and UV photoprotection in transgenic and mutant Petunia leaves by Ken G Ryan; Ewald E Swinny; Kenneth R Markham; Chris Winefield (23-32).
The effects of UVB radiation on plant growth rate, gene expression and flavonoid content in wild-type, and in transgenic and mutant F3′H deficient Petunia lines have been studied for the first time. In wild-type Petunia, UVB induced an increase in total levels of flavonols and this was due to an up-regulation of several genes in the phenylpropanoid pathway. Furthermore, UVB induced a higher rate of production of dihydroxylated flavonols than mono-hydroxylated equivalents. Thus, the ratio of quercetin (ortho-dihydroxylated) to kaempferol (monohydroxylated) increased. In the F3H deficient mutant line, increasing UVB resulted in up-regulation of all of the basic flavonoid biosynthetic genes. Total flavonoids increased to levels significantly higher than in control plants, and the predominant flavonoid was kaempferol. The leaves of these plants grew at a significantly slower rate than comparably treated wild-type plants under ambient or enhanced UVB radiation. This suggests that the predominance of quercetin in the wild-type confers a protective advantage that is not matched in the mutant, even with higher overall flavonoid levels. In contrast, the antisense F3H construct produced an unexpected down-regulation of C4H, CHS and CHI transcription. This resulted in lower total flavonoid production in these plants. The growth rate of these plants was not impaired in UVB to a statistically significant extent, and the Q:K ratio did not change with increasing UVB radiation. This investigation has established a likely correlation between the effect of UVB on plant growth rate, the level of activity of the F3′H gene, and the proposed photoprotection afforded by an increased Q:K ratio.Transgenic and mutant Petunia provide a gradient of F3′H removal to establish a likely correlation between the effect of UVB on plant growth rate, the level of activity of the F3′H gene, and the proposed photoprotection afforded by an increased Q:K ratio.
Keywords: Petunia axillaris × (P. axillaris × P. hybrida); Solanaceae; Mitchell Petunia; UVB; Ultraviolet; Transgenic; Mutant; Flavonoids; Kaempferol; Quercetin; Photoprotection;

Temperature effect on a high stearic acid sunflower mutant by Valle Fernández-Moya; Enrique Martı́nez-Force; Rafael Garcés (33-37).
Vegetable oil with elevated saturated fatty acid content may be useful for producing solid fat without hydrogenation or transesterification. Under the nutritional point of view stearic acid is preferred to other saturated fatty acids because of its neutral effect on serum cholesterol lipoproteins. Selection of a very high stearic acid sunflower (Helianthus annuus L.) line (CAS-14), with up to a 37.3% of stearic acid in the seed oil, and the relationship between the expression of this character and the growth temperature are presented. The mutant was selected from the M2 progeny of 3000 mutagenized seeds (4 mM sodium azide mutagenesis treatment) by analysing the fatty acid composition of half-seed by gas liquid chromatography. In order to genetically fix the mutant character, plants were grown at high day/night temperatures during seed formation. We found that temperatures higher than 30/20 °C are required for good expression of the phenotype, the maximum stearic acid content being obtained at 39/24 °C. This behaviour is totally opposed to that observed in normal and previously isolated high-stearic acid sunflower lines that contain more stearic acid at low temperature. Thus, a new type of temperature regulation on the stearate desaturation must occur. This line is the sunflower mutant with the highest stearic acid content reported so far.The desaturation of stearic acid to oleic acid is reduced by high growth temperature in a sunflower mutant increasing the stearic acid content up to 37.3% in the seed storage lipids.
Keywords: Helianthus annuus; Asteraceae; Sunflower; Temperature; Mutant; Fatty acid desaturation; Lipid; Stearic acid;

Six new partheniol metabolites were isolated from the biotransformation reaction with Mucor circinelloides ATCC 15242. These metabolites are: humula-1(10), 4, 7-trien-6α-ol 2, maali-3-en-8α-ol 3, aromadendrane-4α, 8α, 10α-triol 4, maaliane-4α, 8α, 9α-triol 5, maaliane-5α, 8α, 9α-triol 6, 5(9), 6-tricyclohumulane-4α, 8α, 10α-triol 7. The structural assignments of these metabolites were made possible by different spectroscopic means.Six partheniol metabolites were isolated from the biotransformation reaction with Mucor circinelloides ATCC 15242. These metabolites are belonging to humulane, maaliane and aromadendrane skeleta. The structural assignments of these metabolites were made possible by different spectroscopic means.
Keywords: Partheniol; Guayulins A and B; Humulane; Maaliane; Aromadendrane; Mucor circinelloides; Biotransformation;

Biosynthesis of anthraquinones in cell cultures of Cinchona ‘Robusta’ proceeds via the methylerythritol 4-phosphate pathway by Ying-Shan Han; Robert van der Heijden; Alfons W.M Lefeber; Cornelis Erkelens; Robert Verpoorte (45-55).
Robustaquinone B was found as a major anthraquinone in cell cultures of Cinchona ‘Robusta’ after treatment with a fungal elicitor. Anthraquinones in Cinchona are considered to be of the Rubia type, i.e. rings A and B are derived from chorismate and α-ketoglutarate, whereas ring C is formed from isopentenyl diphosphate (IPP). To determine the origin of IPP, either formed via the mevalonic acid pathway or the 2-C-methyl-d-erythritol 4-phosphate pathway, the incorporation of [1-13C]glucose into robustaquinone B was studied. The 13C labeling of robustaquinone B was analyzed by one- and two-dimensional NMR spectroscopy and the labeling pattern was compared with the hypothetical labeling patterns obtained via the different biosynthetic pathways. The results clearly show that the IPP, constituting the ring C of robustaquinone B, is biosynthesized via the 2-C-methyl-d-erythritol 4-phosphate pathway. Moreover, the data also confirm that rings A and B of robustaquinone B are formed from chorismate and α-ketoglutarate via o-succinylbenzoate.The biosynthetic pathway of anthraquinones was investigated by feeding of [1-13C]glucose to Cinchona ‘Robusta’ cell cultures. The isopentenyl diphosphate unit, which constitutes the C-ring of robustaquinone B, is biosynthesized via the 2-C-methyl-d-erthritol 4-phosphate (MEP) pathway.
Keywords: Cinchona ‘Robusta’; Rubiaceae; Anthraquinone; Isopentenyl diphosphate; Mevalonic acid; 2-C-Methyl-d-erythritol 4-phosphate; NMR spectroscopy;

Incubation of stemodin (1) in cultures of Aspergillus niger ATCC 9142 resulted in the production of 2α,3β,13-trihydroxystemodane (2), 2α,7β,13-trihydroxystemodane (3) and 2α,13,16β-trihydroxystemodane (4), while stemodinone (5) afforded 13,18-dihydroxystemodan-2-one (6) and 13,16β-dihydroxystemodan-2-one (7). Four novel metabolites were obtained from the bioconversion of stemarin (8) by the fungus, namely 18-hydroxystemaran-19-oic acid (9), 7β,18-dihydroxystemaran-19-oic acid (10), 7α,18,19-trihydroxystemarane (11) and 1β-hydroxystemaran-19-oic acid (12). 19-N,N-Dimethylcarbamoxy-13-hydroxystemarane (13) was also transformed to afford 19-N,N-dimethylcarbamoxy-13,17ξ,18-trihydroxystemarane (14).Stemodin and stemodinone were transformed by Aspergillus niger to afford 3 and 2 metabolites, respectively. Stemarin and its dimethyl-carbamate derivative were converted into 4 and 1 products, respectively.
Keywords: Aspergillus niger ATCC 9142; Biotransformation; Stemodane; Stemarane; Diterpene; Stemodia maritima; Hydroxylation;

Responses of anthocyanin-producing and non-producing cells of Glehnia littoralis to radical generators by Yoshie Kitamura; Mina Ohta; Toshihiko Ikenaga; Masami Watanabe (63-68).
Non-anthocyanin-producing cells of Glehnia littoralis produced bergapten following treatment with H2O2 and X-ray, but not with AAPH, whereas the anthocyanin-producing cells did not form bergapten under any of these treatments.The responses of anthocyanin-producing (violet) and non-producing (white) cells of Glehnia littoralis to radical generators were compared. Cell growth, anthocyanin content, phenylalanine ammonia-lyase (PAL) activity and furanocoumarin production were determined after treatment with H2O2, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), X-ray and yeast extract, independently. AAPH and H2O2 repressed the growth of both violet and white cells, but violet cells grew better than white cells. On the other hand, the anthocyanin content in violet cells decreased. Neither X-ray nor yeast extract affected cell growth or pigment production. Treatment with H2O2, yeast extract, and X-ray, but not AAPH, induced PAL activity and furanocoumarin production in white cell cultures, whereas violet cell cultures did not produce furanocoumarin following any of the treatment employed.
Keywords: Glehnia littoralis; Apiaceae; Cell culture; Radical generator; H2O2; 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH); X-ray; Phenylalanine ammonia-lyase (PAL); Anthocyanin; Furanocoumarin;

An insecticidal mixture of tetramethylcyclohexenedione isomers from Kunzea ambigua and Kunzea baxterii by Bhupinder P.S Khambay; David G Beddie; Monique S.J Simmonds (69-71).
A mixture of isomers, all 4-[1-(5,7-dihydroxy-6-methyl-4-oxo-2-phenyl-chroman-8-yl)-3-methyl-butyl]-5-hydroxy-2,2,6,6-tetramethyl-cyclohex-4-en-1,3-diones, which comprises a pair of epimers, each of which is a pair of conformers, has been isolated from the hexane extract of the aerial parts of Kunzea ambigua and K. baxterii (Myrtaceae). The mixture exhibits moderate insecticidal activity in comparison with natural pyrethrum extract.Two Australasian species of Myrtaceae contain a mixture, which has insecticidal activity. It comprises two epimers (at C-1′) each of which exists as a pair of conformers.
Keywords: Myrtaceae; Kunzea; Tetramethylcyclohexenedione; Flavanone; Insecticide;

Antimicrobial constituents from the stem bark of Feronia limonia by Md.Mukhlesur Rahman; Alexander I Gray (73-77).
The stem bark of Feronia limonia (Fam. Rutaceae) yielded (−)-(2S)-5,3′-dihydroxy-4′-methoxy-6″,6″-dimethylchromeno-(7,8,2″,3″)-flavanone along with several known compounds including an alkaloid, five coumarins, a flavanone, a lignan, three sterols and a triterpene. The structures of these compounds were determined by spectroscopic methods, mainly 1D and 2D NMR. The antimicrobial screening of compounds by a microdilution technique resulted in MICs in the range 25–100 μg/ml. Other biological activities of the known compounds are also discussed.A pyranoflavanone, (−)-(2S)-5,3′-dihydroxy-4′-methoxy-6″,6″-dimethylchromeno(7,8,2″,3″)-flavanone, together with 12 known compounds was isolated from the stem bark of Feronia limonia. The antimicrobial activities of these compounds were investigated and other biological activities of the known compounds are discussed.
Keywords: Feronia limonia; Rutaceae; Alkaloid; Coumarins; Flavanones; Lignan; (−)-(2S)-5,3′-Dihydroxy-4′-methoxy-6″,6″-dimethylchromeno(7,8,2″,3″)-flavanone; Antimicrobial;

Bioactive steroidal alkaloid glycosides from Solanum aculeastrum by Alphonse W Wanyonyi; Sumesh C Chhabra; Gerald Mkoji; Udo Eilert; Wilson M Njue (79-84).
Steroidal alkaloid glycosides, solaculine A, and solamargine and β-solamarine were isolated from the root bark and berries, respectively, of Solanum aculeastrum Dunal. Their structures were established by analysis of chemical and spectral evidence. Solamagine and β-solamarine showed molluscicidal activity. Solanum aculeastrum Dunal was investigated for the presence of molluscicidal compounds. This led to the isolation of solaculine A, from the root bark in addition to known steroidal alkaloids; solamargine and β-solamarine from the berries. The structures were elucidated by spectroscopic techniques. Molluscicidal activity of the aqueous extracts of the berries and root bark, and the isolated compounds were investigated.
Keywords: Solanum aculeastrum; Solanaceae; Berries; Root bark; Steroidal alkaloid glycosides; Solamargine; Solamarine; Solaculine A; Molluscicidal activity;

Immunosuppressive constituents from Saussurea medusa by Hongquan Duan; Yoshihisa Takaishi; Hiroshi Momota; Yasukazu Ohmoto; Takao Taki (85-90).
The methanol extract of Saussurea medusa Maxim afforded two lignans: 2α-guaicyl-4-oxo-6α-catechyl-3,7-dioxabicyclo [3.3.0]octane and 1α-hydroxy-2α,4α-guaicyl-3,7-dioxabicyclo[3.3.0]octane; two chlorophyll derivatives: 13-epi-phaeophorbide-a and 13-epi-phaeophorbide-a methyl ester; one megastigmane derivative: 3β-hydroxy-5α,6α-epoxy-7-megastigmen-9-one, along with 19 known compounds. Their structures were established on the basis of spectroscopic studies.Five new and 19 known compounds were isolated from the methanol extract of Saussurea medusa Maxim. Their structures were determined by spectroscopic means. Two lignans showed a significant inhibitory effect on cytokine production.
Keywords: Saussurea medusa; Compositae; Lignan; Phaeophorbide; Immunosuppressive activity;

Tetrahydroisoquinoline-monoterpene glycosides from Cephaelis acuminata by Atsuko Itoh; Yoshikazu Baba; Takao Tanahashi; Naotaka Nagakura (91-97).
From the dried roots of Cephaelis acuminata, five tetrahydroisoquinoline-monoterpene glycosides, 2-O-β-d-glucopyranosyldemethylalangiside, demethylisoalangiside, 6″-O-β-d-glucopyranosylipecoside, 6″-O-α-d-glucopyranosylipecoside and (4R)-4-hydroxyipecoside, were isolated. The structures of these glycosides were determined by spectroscopic and chemical means.From the dried roots of Cephaelis acuminata, five tetrahydroisoquinoline-monoterpene glycosides 1–5 were isolated. The structures of these glycosides were determined by spectroscopic and chemical means.
Keywords: Cephaelis acuminata; Rubiaceae; Roots; Structure elucidation; Tetrahydroisoquinoline-monoterpene glycosides;

The dichloromethane extract of the aerial parts of Artabotrys hexapetalus afforded three β-methoxy-γ-methylene-α,β-unsaturated-γ-butyrolactones, which are proposed to be derived from a C18 unsaturated fatty acid by a biosynthetic route similar to that proposed for the Annonaceous acetogenins. The structure of the unique β-methoxy-γ-methylene-substituted, α,β-unsaturated-γ-butyrolactone ring of artapetalins A–C (13) was determined by 2D-NMR spectroscopic analyses. Two unusual simple butyrolactones, (+)-tulipalin B and (2R,3R)- 3-hydroxy-2-methylbutyrolactone were also isolated from this species.Artapetalins A–C from Artabotrys hexapetalus (Annonaceae) contain an uniquely-substituted butyrolactone ring which is proposed to be biosynthesised by a pathway similar to that for other Annonaceous acetogenins.
Keywords: Artabotrys hexapetalus; Annonaceae; β-Methoxy-γ-methylene-α,β-unsaturated-γ-butyrolactones; Artapetalins A–C, (+)-Tulipalin B; Unsaturated fatty acid derivative; Biosynthesis; 2D NMR;

Constituents of Lepidium meyenii ‘maca’ by Ilias Muhammad; Jianping Zhao; D.Chuck Dunbar; Ikhlas A. Khan (105-110).
The tubers of Lepidium meyenii contain the benzylated derivative of 1,2-dihydro-N-hydroxypyridine, named macaridine, together with the benzylated alkamides (macamides), N-benzyl-5-oxo-6E,8E-octadecadienamide and N-benzylhexadecanamide, as well as the acyclic keto acid, 5-oxo-6E,8E-octadecadienoic acid. The structure elucidation of the isolated compounds was based primarily on 1D and 2D NMR spectroscopic analyses, including 1H–1H COSY, 1H–13C HMQC, 1H–13C HMBC and 1H–1H NOESY experiments, as well as from 1H–15N NMR HMBC correlations for macaridine and N-benzylhexadecanamide.The tubers of Lepidium meyenii yielded the 1,2-dihydro-N-hydroxypyridine derivative, macaridine, and the macamides, N-benzyl-5-oxo-6E,8E-octadecadienamide and N-benzylhexadecanamide, as well as 5-oxo-6E,8E-octadecadienoic acid. The structure elucidation of the isolated compounds was based primarily on 2D NMR analyses, including 15N NMR HMBC experiments.
Keywords: Lepidium meyenii; Brassicaceae; 1,2-Dihydro-N-hydroxypyridine; Macaridine; Macamide; N-Benzyl-5-oxo-6E,8E-octadecadienamide; N-Benzylhexadecanamide; 5-Oxo-6E,8E-octadecadienoic acid; 2D NMR; 1H–15N NMR;

C15 Acetogenins from the red alga Laurencia obtusa by Dimitra Iliopoulou; Constantinos Vagias; Catherine Harvala; Vassilios Roussis (111-116).
Four C15 acetogenins, 13-epilaurencienyne (3Z) (1), 13-epipinnatifidenyne (3E) (2), (3E, 6S , 7R , 9S , 10S , 12R )-9-chloro-13-bromo-6:12-epoxy-7, 10-diacetoxypentadec-3-en-1-yne (3), (3Z, 6S , 7R , 9S , 10S , 12R )-9-chloro-13-bromo-6:12-epoxy-7, 10-diacetoxypentadec-3-en-1-yne (4), along with the known 13-epilaurencienyne (3E) (5), have been isolated from the organic extract of the red alga Laurencia obtusa, collected in the Aegean Sea, Greece. The structures of the new natural products, as well as their relative stereochemistry, were established by means of spectral data analysis, including 2D NMR spectroscopic experiments. Some of the new metabolites exhibited significant insecticidal activity.Four C15 acetogenins 14 have been isolated from the organic extract of the red alga Laurencia obtusa. Their structures, as well as their relative stereochemistry, were established by means of spectral data analysis, including 2D NMR experiments.
Keywords: Laurencia obtusa; Rhodomelaceae; Rhodophyta; Acetogenin; Vinyl acetylene; Cyclic ether; Insecticidal activity;