Phytochemistry (v.65, #2)

Graphical Abstracts (143-145).

Eicosapentaenoic acid (20:5Δ5,8,11,14,18; EPA) is important in human health and nutrition, as a precursor for anti-inflammatory eicosanoids and membrane component. EPA is thought to have a protective role against cardiovascular disease and Metabolic Syndrome. EPA is currently obtained from fish oils, a diminishing resource. The biosynthesis of EPA is described, as is the potential for production in transgenic plants.Long chain polyunsaturated fatty acids are now known to play important roles in human health. In particular, eicosapentaenoic acid (20:5Δ5,8,11,14,17; n-3: EPA) is implicated as a protective agent in a range of pathologies such as cardiovascular disease and Metabolic Syndrome (Syndrome X). Eicosapentaenoic acid is currently sourced from fish oils, the presence of this fatty acid being due to the dietary piscine consumption of EPA-synthesising micro-algae. The biosynthetic pathway of EPA has been elucidated, and contains several alternative metabolic routes. Progress in using “reverse engineering” to transgenically mobilize the trait(s) for EPA are considered. In particular, the prospect of producing this important polyunsaturated fatty acid in transgenic oilseeds is highlighted, as is the urgent need for a sustainable replacement for diminishing fish stocks.
Keywords: Polyunsaturated fatty acids; Eicosapentaenoic acid; Metabolic engineering; Metabolic Syndrome; Plant biotechnology;

cis-3-Hexenal production in tobacco is stimulated by 16-carbon monounsaturated fatty acids by Mei Hong; Barbara A Zilinskas; Douglas C Knipple; Chee-Kok Chin (159-168).
Transgenic tobacco plants O9 and T16 expressing the yeast acyl-CoA Δ9 desaturase and an insect acyl-CoA Δ11 desaturase, respectively, displayed altered profiles of fatty acids compared to wild-type tobacco plants and marked increases in cis-3-hexenal, a major leaf volatile derived from α-linolenic acid (18:3). As expected, O9 and T16 plants had increased levels of the major unsaturated fatty acid products formed by the transgenic desaturases they expressed, viz., palmitoleic acid (16:1Δ9) and palmitvaccenic acid (16:1Δ11), respectively. In addition, levels of 18:3 lipid declined slightly and the pool of free 18:3, which accounts for about 30% of free fatty acids in wild-type plants, disappeared completely in both transgenics. Both O9 and T16 plants were found to have a two-fold increase in 13-lipoxygenase (13-LOX) activity, which catalyzes the first of two steps leading to hexenal production from 18:3. In O9 and T16 plants, the activity of 9-lipoxygenase and hydroperoxide lyase, the latter catalyzing the formation of cis-3-hexenal from α-linolenic acid hydroperoxide, was significantly different from that of the wild-type plants. Although 16:1Δ9 and 16:1Δ11 had no direct effects on 13-LOX activity in vitro, cis-3-hexenal production increased in tobacco leaves treated with these fatty acids, suggesting that they may act in vivo by stimulating 13-LOX gene expression.Tobacco plants expressing the yeast Δ9 desaturase gene and an insect Δ11 desaturase gene, respectively, produced elevated levels of cis-3-hexenal.
Keywords: Cis-3-hexenal; Palmitoleic acid; Palmitraccenic acid; Hexanal; Lipoxygenase;

Agrobacterium tumefaciens AK-6b gene modulates phenolic compound metabolism in tobacco by Ivan Gális; Yasutaka Kakiuchi; Petr Šimek; Hiroetsu Wabiko (169-179).
The 6b gene (AK-6b) of Agrobacterium tumefaciens AKE10 can substitute for the requirement of tobacco tissues for auxin and cytokinin to maintain callus growth in the culture medium. To identify compounds that might be involved in this process we analyzed phenolic metabolites in transgenic tobacco tissues expressing the AK-6b gene. On medium containing both cytokinin and auxin (SH medium), transgenic calli accumulated higher levels of chlorogenic acid, caffeoyl putrescine, rutin and kaempferol-3-rutinoside, than did wild-type tissues. In contrast, the levels of scopolin and its aglycone, scopoletin were lower in transgenic tissues. On hormone-free medium, these phenolic compounds showed neither significant levels nor an apparent relationship with AK-6b transcript levels, except for the negatively correlated levels of scopoletin and AK-6b transcripts. Apparently, the AK-6b gene acts, in SH medium, to redirect the synthesis of scopolin in tobacco tissues towards the preferential synthesis of caffeic acid derivatives and flavonoids.Plant tumourigenic AK-6b gene directs the synthesis of flavenoids including rutin in tobacco
Keywords: Nicotiana tabacum; Solanaceae; Tobacco; Metabolism; Phenylpropanoids; Flavonoids; Chlorogenic acid; Caffeoylputrescine; Rutin; Kaempferol-3-rutinoside; Scopolin; Scopoletin;

β-Adenosine, a bioactive compound in grass chaff stimulating mushroom production by Denisa L. Domondon; Weidong He; Norbert De Kimpe; Monica Höfte; Joseph Poppe (181-187).
Fructification and yield of the edible mushrooms Pleurotus pulmonarius and Stropharia rugosoannulata are clearly enhanced when wheat straw is supplemented with 30% Lolium perenne grass chaff. The bioactive compound in the methanol extract of grass chaff was identified as β-adenosine. In vitro biological activity tests showed that 0.012 mg of β-adenosine per ml of medium stimulated earlier fructification of Pleurotus pulmonarius. Mushroom fruiting trials showed that when 12 mg β-adenosine was added to 1 kg wet wheat straw, primordia of Pleurotus pulmonarius appeared two days earlier and primordia of Stropharia rugosoannulata appeared 18 days earlier when compared to pure wheat straw substrate. This concentration of β-adenosine had no impact on the mushroom yield of Pleurotus, but resulted in a 2.2 fold increase in yield for Stropharia. β-Adenosine at 25 mg per kg wet wheat straw increased the yield of Pleurotus with 52% and the yield of Stropharia with 258%, but this concentration delayed primordial formation in Pleurotus.Supplementation of wheat straw with 30% Lolium perenne grass chaff enhances the fructification and yield of the edible mushrooms Pleurotus pulmonarius and Stropharia rugosoannulata. We have identified β-adenosine as one of the bioactive compounds in grass chaff that stimulates fruit body formation and increases yield.
Keywords: Lolium perenne; Pleurotus pulmonarius; Stropharia rugosoannulata; Mushroom fruit body formation; Oyster mushroom; King Stropharia; Rye grass;

Six forms of sphaeropsidins (SA–SF), three- and tetra-cyclic unrearranged pimarane diterpenes produced by Sphaeropsis sapinea f. sp. cupressi, as well as eight derivatives obtained by chemical modification of SA–SC, were assayed for their bioactivity. The effect of each compound on plants which are host or non-host of the pathogen was investigated. Activity on some plant pathogenic fungi was also tested. Some structure–activity relationships have been identified for both phytotoxic and antifungal activity. It appears that the integrity of the tricyclic pimarane system, the preservation of the double bond C(8)–C(14), the tertiary hydroxyl group at C-9, the vinyl group at C-13, and the carboxylic group at C-10 as well as the integrity of the A-ring provide these molecules with non selective phytotoxic and antimycotic activity.We report the structure–activity relationship of sphaeropsidins A (1)–F, primarane diterpenes produced by Sphaeropsis sapinea f. sp. cupressi, the casual agent of a canker form on cypress tree.
Keywords: Cypress; Cupressaceae; Sphaeropsis sapinea f. sp. cupressi; Cypress canker disease; Pimarane diterpene; Sphaeropsidin derivatives; Structure–activity relationship;

Sesquiterpenes of the liverwort Scapania undulata by Adewale Martins Adio; Claudia Paul; Petra Kloth; Wilfried A König (199-206).
The essential oil of the liverwort Scapania undulata, collected in the Harz mountains, Northern Germany, was analysed by gas chromatography (GC), GC–mass spectrometry (MS) and several new components were isolated and investigated by various NMR techniques. As new natural compounds the sesquiterpene hydrocarbons (+)-helminthogermacrene (1) [the 4Z-isomer of germacrene A (9)], (−)-cis-β-elemene (2) as a Cope-rearrangement product of 1, (+)-β-isolongibornene (3) and (−)-perfora-1,7-diene (4) could be identified. 1 has an identical mass spectrum and identical GC retention time on a non-polar stationary phase as germacrene A (9) but is considerably more stable than the latter. The Cope-rearrangement of 1 proceeds slowly at 350 °C and (−)-cis-β-elemene (2) is formed together with small amounts of other diastereoisomers.The volatile sesquiterpene constituents of the liverwort Scapania undulata (longiborneol chemotype) were investigated by spectroscopic methods. (+)-Helminthogermacrene, (−)-cis-β-elemene, (+)-β-isolongibornene (3) and (−)-perfora-1,7-diene (4) were identified as new natural products.
Keywords: Scapania undulata; Liverworts; Sesquiterpene hydrocarbons; Cope rearrangement; (+)-Helminthogermacrene; (+)-β-Isolongibornene; (−)-cis-β-Elemene; (−)-Perfora-1,7-diene;

Neolignan and flavonoid glycosides in Juniperus communis var. depressa by Tsutomu Nakanishi; Naoki Iida; Yuka Inatomi; Hiroko Murata; Akira Inada; Jin Murata; Frank A. Lang; Munekazu Iinuma; Toshiyuki Tanaka (207-213).
Two neolignan glycosides (junipercomnosides A and B) were isolated from aerial parts of Juniperus communis var. depressa along with two known neolignan glycosides and seven flavonoid glycosides. The structures of the isolated compounds were determined by spectral analysis, in particular by 2D-NMR analysis. The significance of distribution of flavonoids in the chemotaxonomy of genus Juniperus was also discussed.Two neolignan glycosides were isolated from Juniperus communis var. depressa along with known neolignan and flavonoid glycosides.
Keywords: Juniperus communis var. depressa; Cupressaceae; Neolignan glycoside; Flavonoid glycoside; Junipercomnoside A; Junipercomnoside B; Isoscutellarein 7-O-β-xylopyranoside; Hypolaetin 7-O-β-xylopyranoside;

Trimeric proteracacinidins and a (6→6)-bis-leucoteracacinidin from Acacia galpinii and Acacia caffra by Linette Bennie; Johan Coetzee; Elfranco Malan; Desmond Slade; Jannie P.J. Marais; Daneel Ferreira (215-220).
The rare series of trimeric proteracacinidins is extended by identification of the first analogs with exclusive C–C interflavanyl bonds, i.e. epioritin-(4β→6)-oritin-(4α→6)-epioritin-4α-ol,oritin-(4β→6)-oritin-(4α→6)-epioritin-4α-ol, and epioritin-(4β→6)-epioritin-(4β→6)-epioritin-4α-ol. These compounds are accompanied by the bis-leucoteracacinidin, epioritin-4α-ol-(6→6)-epioritin-4β-ol, the first naturally occurring bis-flavan-3,4-diol.The rare series of trimeric proteracacinidins is extended by identification of the first analogues with exclusive C–C interflavanyl bonds, i.e. the epioritin-(4β→6)-oritin-(4α→6)-epioritin-4α-ol. These compounds are accompanied by the first bis-flavan-3,4-diol.
Keywords: Acacia galpinii; Acacia caffra; Leguminosae; Trimeric proteracacinidins; Proanthocyanidins; Bis-flavan-3,4-diol;

Prenylated flavonoids, monoterpenoid furanocoumarins and other constituents from the twigs of Dorstenia elliptica (Moraceae) by Berhanu M. Abegaz; Bonaventure T. Ngadjui; Gabriel N. Folefoc; Serge Fotso; Pantaleon Ambassa; Merhatibeb Bezabih; Etienne Dongo; Frode Rise; Dirk Petersen (221-226).
A new prenylated flavonoid and two monoterpenoid substituted coumarins were identified from the twigs of Dorstenia elliptica (Moraceae)A monoprenylated flavan and two monoterpenoid substituted furanocoumarins were isolated from the twigs of Dorstenia elliptica along with 3-(3,3-dimethylallyl)-4,2′,4′-trihydroxylchalcone, psoralen, bergapten, O-[3-(2,2-dimethyl-3-oxo-2H-furan-5-yl)butyl]bergaptol, β-sitosterol and its β-d-glucopyranoside. The structure of the flavan was determined as 6(1,1-dimethylallyl)-7,4′-dihydroxylflavan and the monoterpenoid substituted furanocoumarins were assigned as O-[3-(2,2-dimethyl-3-oxo-2H-furan-5-yl)-3-hydroxybutyl]-bergaptol and O-[2-(5-hydroxy-2,6,6-trimethyl-3-oxo-2H-pyran-2-yl)ethyl]bergaptol, respectively, using spectroscopic analysis, especially, 2D NMR spectra.
Keywords: Dorstenia elliptica; Moraceae; Twigs; Isolation; 6(1,1-dimethylallyl)-7,4′-dihydroxylflavan; Monoterpenoid furanocoumarins;

The caulindoles: dimeric prenylindoles from Isolona cauliflora by John J. Makangara; Leonia Henry; Stephan A. Jonker; Mayunga H.H. Nkunya (227-232).
Four dimeric prenylindoles occurring in diastereomeric pairs, the caulindole A–D, 5-(3-methyl-2-butenyl)-1H-indole, and (E)-5-(3-methylbuta-1,3-dienyl)-1H-indole were isolated from the stem and root barks of Isolona cauliflora, an ecologically endangered Annonaceae species. Structural determination was achieved based on interpretation of spectroscopic data. Biogenetically, the caulindoles are considered as Diels–Alder-type cycloaddition products of mono- and/or bis-prenylindoles [e.g. 5-(3-methyl-2-butenyl)-1H-indole and (E)-5-(3-methylbuta-1,3-dienyl)-1H-indole] as the dienes and dienophiles.Two diastereomeric pairs of dimeric prenylindoles, the caulindoles A–D (1–4), 5-(3-methyl-2-butenyl)-1H-indole, and (E)-5-(3-methylbuta-1,3-dienyl)-1H-indole were isolated from the ecologically endangered Annonaceae species, Isolona cauliflora and their structures determined from spectroscopic data. Biogenetically, the caulindoles may be Diels–Alder-type cycloaddition products of mono-prenylindoles as the dienes and dienophiles.
Keywords: Isolona cauliflora; Annonaceae; Prenylindoles; Caulindoles; Antifungals;

Rapid dereplication of estrogenic compounds in pomegranate (Punica granatum) using on-line biochemical detection coupled to mass spectrometry by Danny A van Elswijk; Uwe P Schobel; Ephraim P Lansky; Hubertus Irth; Jan van der Greef (233-241).
During recent years, phytoestrogens have been receiving an increasing amount of interest, as several lines of evidence suggest a possible role in preventing a range of diseases, including the hormonally dependent cancers. In this context, various parts of the pomegranate fruit (Punica granatum; Punicaceae), e.g. seed oil, juice, fermented juice and peel extract, have been shown to exert suppressive effects on human breast cancer cells in vitro. On-line biochemical detection coupled to mass spectrometry (LC-BCD–MS) was applied to rapidly profile the estrogenic activity in the pomegranate peel extract. The crude mixture was separated by HPLC, after which the presence of biologically active compounds, known or unknown, was detected by means of an on-line β-estrogen receptor (ER) bioassay. Chemical information, such as molecular weight and MS/MS fingerprint, was obtained in real time by directing part of the HPLC effluent towards a mass spectrometer. Using this approach in total three estrogenic compounds, i.e. luteolin, quercetin and kaempferol, were detected and identified by comparing the obtained molecular weights and negative ion APCI MS/MS spectra with the data of an estrogenic compound library. Although well known in literature and widely distributed in nature, the presence of these phytoestrogenic compounds in pomegranate peel extract was not reported previously. Compared to traditional screening approaches of complex mixtures, often characterized by a repeating cycle of HPLC fractionation and biological screening, LC-BCD–MS was shown to profoundly accelerate the time required for compound description and identification.Bioactive compounds, such as kaempferol (see displayed structure) are rapidly detected and identified in compex mixtures such as natural product extracts.
Keywords: Pomegranate; Punica granatum; On-line biochemical detection–mass spectrometry; Phytoestrogens;