Phytochemistry (v.65, #15)

Polyphenol oxidase expression in potato (Solanum tuberosum) tubers inhibited to sprouting by treatment with iodine atmosphere by Francesco Eolini; Alejandro Hochkoeppler; Andrea Credi; Antonio Gonzàlez Vara Y Rodrı́guez; ValeriaPoggi (2181-2187).
In bud tissues of potatoes exposed to iodine atmosphere the transcription of polyphenol oxidases features an increase followed by a decrease occurring simultaneously with the suppression of sprouting.Iodine-saturated atmosphere was found to inhibit the sprouting of potato (Solanum tuberosum L.) tubers. The iodine concentration in tuber tissues increased as a function of exposure length, and the onset of inhibition of sprouting was found to depend on tubers genotype. During the time-course of the treatment, the transcription of polyphenol oxidases (EC and EC was undetectable in tuber peel, whereas in bud tissues featured an increase, followed by a decrease occurring simultaneously with the suppression of sprouting. The treatment of tubers with iodine strongly affected the expression of polyphenol oxidases at the transcriptional level. Polyphenol oxidase activity in buds poorly reflected the corresponding level of transcription; similarly, little differences were found among the enzyme isoforms expressed in buds as a function of length of exposure to iodine. These findings suggest that the induction of polyphenol oxidases mRNAs transcription could probe the inhibition of sprouting by iodine.
Keywords: Solanum tuberosum; Solanaceae; Potato tuber; Enzyme expression; Polyphenol oxidase; Inhibition of sprouting; Iodine;

Expression of a Stokesia laevis epoxygenase gene by Tomoko Hatanaka; Rena Shimizu; David Hildebrand (2189-2196).
A cDNA cloned from Stokesia laevis encodes an epoxygenase active in yeast and Arabidopsis.Epoxy fatty acids have a number of important uses and there is interest in enzymes catalyzing their synthesis from renewable sources. Both cytochrome P450 monooxygenases and divergent forms of di-iron desaturases are known to produce epoxy fatty acids in plants. Degenerate primers based on conserved sequences of Δ12 desaturase-like genes led to the isolation of an epoxygenase gene from Stokesia laevis. The cDNA is 1.4 kb and it encodes 378 amino acids. The similarities of this gene at the amino acid sequence level with epoxygenases of Vernonia and Crepis, and the Δ12 desaturases of soybean, FAD2-1 and FAD2-2, are 84%, 69%, 49%, and 55%, respectively. When the vector, pYES2, was used to transform yeast, epoxy fatty acid formation was observed in the cells. The effects of electron donors in the yeast expression system were tested but cytochrome b5 and cytochrome b5 reductase genes from Arabidopsis thaliana co-expressed with the epoxygenase had little effect on vernolic acid accumulation in the yeast. Finally, this gene, driven by a seed-specific phaseolin promoter, was cloned into a TDNA-vector and transferred into Arabidopsis plants. The results showed that T2 seeds of transgenic Arabidopsis expressing the Stokesia gene accumulated vernolic acid but no vernolic acid was detected in control plants. Northern blot analysis indicates this S. laevis epoxygenase gene is expressed mainly in developing seeds and no transcript was detected in leaves or roots.
Keywords: Stokesia laevis; Asteraceae; Epoxygenase; Vernolic acid;

Effects of elevated CO2 on the vasculature and phenolic secondary metabolism of Plantago maritima by Matthew P. Davey; David N. Bryant; Ian Cummins; Trevor W. Ashenden; Phillip Gates; Robert Baxter; Robert Edwards (2197-2204).
A one-year exposure to elevated atmospheric CO2 resulted in modifications of the vasculature and lignification in Plantago maritima. An accumulation of caffeic acid also occurred in the shoots, while in the roots p-coumaric acid and verbascoside content was enhanced by elevated CO2.We have examined the effect of elevated CO2 on the vasculature and phenolic secondary metabolism on clones of the maritime plant Plantago maritima (L.). Plants were exposed to either ambient (360 μmol CO2 mol−1) or elevated (600 μmol CO2 mol−1) atmospheric CO2 within a Solardome facility and harvested after 12 months' growth. Histochemical analysis of the leaves identified increases in the diameter of the minor leaf vein and associated lignified vessels in plants exposed to elevated CO2. In the roots the number of lignified root vessels and stele width were also increased, but overall the lignified vessel-wall thickness was reduced in plants exposed to elevated CO2, compared to those grown under ambient CO2. To investigate whether or not these subtle changes in lignification were associated with perturbations in phenolic metabolism, aromatic natural products were analysed by HPLC–MS after treatment with cellulase to hydrolyse the respective glycosidic conjugates. The phenylpropanoids p-coumaric acid, caffeic acid, ferulic acid and the flavone luteolin were identified, together with the caffeoyl phenylethanoid glycosides, verbascoside and plantamajoside which were resistant to enzymatic digestion. Exposure to enhanced CO2 resulted in subtle changes in the levels of individual metabolites. In the foliage a one-year exposure to enhanced CO2 resulted in an increased accumulation of caffeic acid, whilst in the roots p-coumaric acid and verbascoside were enhanced. Our results suggest that significant changes in the vasculature of P. maritima on exposure to increased CO2 are associated with only minor changes in the leaves of specific lignin-related metabolites.
Keywords: Plantago maritima; Plantaginaceae; Elevated atmospheric CO2; Phenylpropanoids; Phenylethanoid glycosides; Flavonoids; Lignification;

Biotransformation of hydrocortisone by a natural isolate of Nostoc muscorum by Mojtaba Tabatabaei Yazdi; Homeira Arabi; Mohammad Ali Faramarzi; Younes Ghasemi; Mohsen Amini; Shadman Shokravi; Farzaneh Aziz Mohseni (2205-2209).
Hydrocortisone (III) was converted in the culture of an isolated strain of the cyanobacterium Nostoc muscorum PTCC 1636 into some androstane and pregnane derivatives. The bioproducts obtained were purified using chromatographic methods and identified as 11β-hydroxytestosterone (I), 11β-hydroxyandrost-4-en-3,17-dione (II) and 11β,17α,20β,21-tetrahydroxypregn-4-en-3-one (IV) on the basis of their spectroscopic features.Hydrocortisone was converted in the culture of an isolated strain of the cyanobacterium Nostoc muscorum PTCC 1636 into some androstane and pregnane derivatives. The microorganism was isolated during a screening program from soil samples collected from paddy fields of north of Iran. The bioproducts obtained were purified using chromatographic methods and identified as 11β-hydroxytestosterone, 11β-hydroxyandrost-4-en-3,17-dione and 11β,17α,20β,21-tetrahydroxypregn-4-en-3-one on the basis of their spectroscopic features.
Keywords: Bioconversion; Hydrocortisone; Microalgae; Cyanobacteria; Nostoc muscorum;

As part of our ongoing programme, eight analogues of the mild antiviral and cytotoxic diterpene stemodin were incubation with Rhizopus oryzae ATCC 11145. The results provide useful information about the relationship of the functional groups of the substrates and their products of bioconversion.Incubation of 2α,13(R)-dihydroxystemodane (3) with Rhizopus oryzae ATCC 11145 gave 2α,7β,13(R)-trihydroxystemodane (11) while biotransformation of 13(R)-hydroxystemodan-2-one (5) yielded 6α,13(R)-dihydroxystemodan-2-one (12) and 7β,13(R)-dihydroxystemodan-2-one (13). Bioconversion of 2β,13(R)-dihydroxystemodane (7) with Rhizopus afforded 2β,7,13(R)-trihydroxystemodane (14). The results complement data from our previous work and provide more information about the effect of functional groups of stemodane substrates on the site of hydroxylation.
Keywords: Rhizopus oryzae ATCC 11145; Stemodia maritima; Scrophulariaceae; Stemodane; Diterpene; Biotransformation; Hydroxylation; Rhizopus arrhizus;

Delphinidin accumulation is associated with abnormal flower development in petunias by Toshio Ando; Motoko Takahashi; Taku Nakajima; Yukiko Toya; Hitoshi Watanabe; Hisashi Kokubun; Fumi Tatsuzawa (2219-2227).
No commercial petunia lines highly accumulate delphinidin 3-glucoside in the in flower. Active flavonoid 3′,5′-hydroxylase (F3′5′H) and inactive anthocyanidin 3-glucoside-rhamnosyltransferase (RT) are considered to be associated with a dull-coloured crumpled corolla-limb.The relative floral anthocyanidin contents of 195 commercial petunias with floral colours other than white and yellow were determined using HPLC, and the presence of five anthocyanidins {cyanidin, peonidin, delphinidin, petunidin, and malvidin} was confirmed. Pelargonidin was not detected, and delphinidin was not a major component. Using a principal component analysis of the relative anthocyanidin contents, the petunias were classified into three phenotype-groups accumulating cyanidin, peonidin, or malvidin, {plus petunidin} as the major anthocyanidin. A fourth phenotype was segregated in the progeny obtained by self-pollinating an F1 hybrid of the malvidin group; this accumulated delphinidin 3-glucoside in a markedly crumpled corolla-limb (delphinidin group). Such inferior floral traits, associated with the accumulation of delphinidin 3-glucoside, are thought to be the driving force that removed the delphinidin group from commercial petunias. A comparison of flowers of the delphinidin group and those of the other groups may provide a useful tool towards a deeper understanding of how anthocyanin biosynthesis relates to normal development of the corolla.
Keywords: Petunia; Solanaceae; Anthocyanin biosynthesis; Crumpled flower; Principal component analysis; Flavonoid 3′,5′-hydroxylase; Anthocyanidin 3-glucoside-rhamnosyltransferase;

Phenyl-terminated fatty acids in seeds of various aroids by Juris Meija; Victor G. Soukup (2229-2237).
Series of homologous saturated and unsaturated ω-phenyl fatty acids were found and characterized in various genera of the subfamily Aroideae of the Araceae.A series of homologous ω-phenylalkanoic acids and ω-phenylalkenoic acids were isolated from seed lipids of various genera of the subfamily Aroideae of Araceae (the Jack-in-the-Pulpit family) and characterized. Besides the major acids, 11-phenylundecanoic acid, 13-phenyltridecanoic acid and 15-phenylpentadecanoic acid, all other homologous odd carbon number ω-phenylalkanoic acids from C7 to C23 were detected in trace amounts. Additionally, one even carbon number acid, 12-phenyldodecanoic acid was found in several specimens in trace amounts. Similarly, two series of homologous odd carbon number monounsaturated ω-phenylalkenoic acids were found and characterized using dimethyl disulfide derivatization to locate the positions of their double bonds. In five acids from C11 to C19, the double bond is located at the same distance, Δ7, from the phenyl ring. In the other two acids of C13 and C15 chain length, the double bond is located at Δ5 from the phenyl ring.
Keywords: Aroids; Aroideae; Araceae; Arisaema; Arum; Typhonium; ω-Phenylalkanoic acids; ω-Phenylalkenoic acids; GC/TOF-MS;

3-Hydroxypropionic acid as a nematicidal principle in endophytic fungi by Michael Schwarz; Bärbel Köpcke; Roland W.S. Weber; Olov Sterner; Heidrun Anke (2239-2245).
3-Hydroxypropionic acid, not previously reported from fungi, is a common nematicidal metabolite among fungal endophytes.3- Hydroxypropionic acid was isolated by bioactivity-guided fractionation of extracts obtained from submerged cultures of several endophytic fungi isolated from above-ground plant organs. This compound showed selective nematicidal activity against the plant-parasitic nematode Meloidogyne incognita with LD50 values of 12.5–15 μg/ml. Activity against the saprophytic Caenorhabditis elegans was fivefold lower. No antimicrobial, cytotoxic or phytotoxic effects were observed. Propionic acid and d- and l-lactic acids were not active against either nematode species. Based on morphological features and ITS, 18S and 28S rDNA analyses, the producing strains were identified as Phomopsis phaseoli isolated from the leaf of a tropical tree, and four strains of Melanconium betulinum isolated from twigs of Betula pendula and B. pubescens in Germany. This is the first report of 3-hydroxypropionic acid in fungi, and of the nematicidal activity of this metabolite.
Keywords: Diaporthe phaseolorum; Phomopsis phaseoli; Melanconis stilbostoma; Melanconium betulinum; Meloidogyne incognita; Endophyte; Nematicidal activity; 3-Hydroxypropionic acid;

Inhibitors of the LPS-induced NF-κB activation from Artemisia sylvatica by Hui Zi Jin; Jeong Hyung Lee; Dongho Lee; Young Soo Hong; Young Ho Kim; Jung Joon Lee (2247-2253).
Three guaianolide type sesquiterpene lactones, 3α,4α-epoxyrupicolins C–E (13), together with six known sesquiterpenes (49) were isolated and identified from the methanol extract of the aerial parts of Artemisia sylvatica, and their biological activities were reported.Three guaianolide sesquiterpene lactones, 3α,4α-epoxyrupicolins C–E, together with six known sesquiterpenes, artemisolide, 3-methoxytanapartholide, deacetyllaurenobiolide, moxartenolide as well as arteminolides B and D were isolated by bioassay-guided fractionation from the methanol extract of the aerial parts of Artemisia sylvatica using the NF-κB mediated reporter gene assay. All isolated compounds displayed inhibitory activity on the LPS-induced NF-κB activation, NO production, and TNF-α production with IC50 values of 0.49–7.17, 1.46–6.16, and 3.19–27.76 μM, respectively, in RAW264.7 cells. It was also established that arteminolide B suppressed the expression of NF-κB target genes such as iNOS and COX-2. This is the first report of NF-κB inhibitory activities of these compounds and supports the pharmacological use of Artemisia sylvatica, which has been employed as an herbal medicine for the treatment of inflammation.
Keywords: Artemisia sylvatica; Compositae; Sesquiterpene lactone; NF-κB; NO; TNF-α; iNOS; COX-2;

Monoamine oxidase inhibitors from Gentiana lutea by Hiroyuki Haraguchi; Yasumasa Tanaka; Amal Kabbash; Toshihiro Fujioka; Takashi Ishizu; Akira Yagi (2255-2260).
A dimeric chalcone derivative (1) and a hydrophobic dihydrocoumarin (3) were newly isolated from Gentiana lutea together with 5-hydroxyflavanone (4) as brain mitochondrial monoamine oxidase inhibitors.Three monoamine oxidase (MAO) inhibitors were isolated from Gentiana lutea. Their structures were elucidated to be 3-3″ linked-(2′-hydroxy-4-O-isoprenylchalcone)-(2‴-hydroxy-4″-O-isoprenyldihydrochalcone) (1), 2-methoxy-3-(1,1′-dimethylallyl)-6a,10a-dihydrobenzo(1,2-c)chroman-6-one and 5-hydroxyflavanone. These compounds, and the hydrolysis product of 1, displayed competitive inhibitory properties against MAO-B which was more effective than MAO-A.
Keywords: Gentiana lutea; Gentianaceae; Monoamine oxidase; 3-3″Linked-(2′-hydroxy-4-O-isoprenylchalcone)-(2‴-hydroxy-4″-O-isoprenyldihydrochalcone); 2-Methoxy-3-(1,1′-dimethylallyl)-6a,10a-dihydrobenzo(1,2-c)chroman-6-one; 5-Hydroxyflavanone;

Isogermacrene A, a proposed intermediate in sesquiterpene biosynthesis by Thomas Hackl; Wilfried A. König; Hermann Muhle (2261-2275).
A sesquiterpene hydrocarbon, isogermacrene A (5), which is structurally related to the gorgonanes and zieranes, was isolated from the essential oil of Saccogyna viticulosa (Hepaticae). This monocyclic compound is proposed as the biogenetic intermediate of some rare sequiterpene skeletons.In the essential oil of the liverwort Saccogyna viticulosa, collected on the island of Madeira, the new sesquiterpene hydrocarbons isogermacrene A (5) and its Cope rearrangement product iso-β-elemene (6) were identified. 5 is proposed to act as the biogenetic precursor of several new sesquiterpenes identified in the volatiles of S. viticulosa. These include iso-α-humulene, α-gorgonene, gorgona-1,4(15),11-triene and gorgon-11-en-4-ol. In addition, the Cope product of zierene, isozierene, allo-aromadendra-4(15),10(14)-diene, aromadendra-4(15),10(14)-dien-1-ol and a prenylguaiane diterpene alcohol, named viticulol, were identified as new natural products.
Keywords: Saccogyna viticulosa; Liverwort; Sesquiterpenes; Isogermacrene A; Iso-β-elemene; Cope rearrangement; Biosynthesis;

Sesquiterpene constituents from the essential oils of the liverworts Mylia taylorii and Mylia nuda by Stephan H. von Reuß; Chia-Li Wu; Hermann Muhle; Wilfried A. König (2277-2291).
Chemical investigation of the essential oils from Mylia taylorii and Mylia nuda resulted in the isolation and structure elucidation of 13 new sesquiterpene constituents including three new carbon skeletons.The essential oils and extracts of Mylia taylorii and M. nuda were investigated by gas chromatography, mass spectrometry, NMR spectroscopy and chemical correlations. Beside several known compounds 13 new constituents including three new carbon skeletons could be identified. Four hydrocarbons with a molecular formula of C15H22 (m/z 202) were identified as myli-4(15)-ene (1), aromadendra-1(10),4(15)-diene (19), aromadendra-4,10(14)-diene (20) and aromadendra-4,9-diene (21). Three oxaspiro-compounds were identified as 7-epi-bourbon-3-en-5,11-oxide (22), guai-3,10(14)-dien-5,11-oxide (23) and guai-3,9-dien-5,11-oxide (24). The absolute configuration of myli-4(15)-en-3-one (5) could be established by chemical correlation. Together with α-taylorione (7) the corresponding 6,11-seco-compound taylopyran (25) with a new carbon skeleton was identified which serves as a precursor to taylocyclane (26) and taylofuran (27). Taynudol (28) contains a new carbon skeleton with a cyclobutenyl structure.
Keywords: Mylia taylorii; Mylia nuda; Liverworts; Sesquiterpenoids; Structure elucidation;

Compound-specific hydrogen isotopic compositions of saturated, monounsaturated and polyunsaturated fatty acids in natural marine macroalgae have been determined in order to clarify hydrogen isotopic fractionations during their desaturation and elongation associated with polyunsaturated fatty acid biosynthesis.Compound-specific hydrogen isotopic compositions (δD) of saturated, monounsaturated and polyunsaturated fatty acids have been determined for natural marine macroalgae including two brown algae (Heterokontophyta) and two red algae (Rhodophyta). δD values of individual fatty acids from four macroalgae exhibit a wide variation ranging from −189‰ to +48‰. Generally, stearic (18:0), arachidic (20:0) and behenic acids (22:0) are much more enriched in D by up to ∼180‰ relative to myristic (14:0), palmitic (16:0), octatetraenoic [18:4(n  − 3)] and eicosapentaenoic acids [20:5(n  − 3)]. Other fatty acids such as oleic [18:1(n  − 9)], lenoleic [18:2(n  − 6)] and linolenic acids [18:3(n  − 3)] fall isotopically between these fatty acids. This wide δD variation of fatty acids is probably explained by the hydrogen isotopic fractionation during desaturation being much larger than that during elongation in the network of polyunsaturated fatty acid biosynthesis. A large hydrogen isotopic fractionation during desaturation may cause D-enrichment in the remaining hydrogen of the residual fatty acids, which could be controlled by the relative flux into their desaturates.
Keywords: Marine macroalgae; Hydrogen isotopic composition; Hydrogen isotopic fractionation; Lipid biosynthesis; Desaturation; Elongation; Polyunsaturated fatty acid;