The Mass Spectra of Phrymarolins, 1,2-Dioxygenated-3,7-dioxabicyclo[3.3.0]octane Lignans

The mass spectra of phrymarolins, which were isolated from Phryma leptostachya L. as novel lignans, have been investigated. The most predominant fragmentation was the splitting of the acetal linkage. The fragment ions (m) and (n), at m/e 141 and m/e 99 respectively, are considered to be characteristic to the lignans. The accurate molecular weight measurements of important ions provided a further evidence for the structure of phrymarolin-I.     

Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures

Lignin and lignans share monolignols as common precursors and are both potentially involved in plant defence against pathogens. In this study, we investigated the effects of fungal elicitors on lignin and lignan metabolism in flax (Linum usitatissimum) cell suspensions. Cell suspension cultures of flax were treated with elicitor preparations made from mycelium extracts of Botrytis cinerea, Phoma exigua and Fusarium oxysporum F ssp lini. Elicitors induced a rapid stimulation of the monolignol pathway, as confirmed by the increase in PAL (phenylalanine ammonia-lyase, EC 4.1.3.5), CCR (cinnamoyl-CoA reductase EC 1.2.1.44) and CAD (cinnamyl alcohol dehydrogenase EC 1.1.1.195) gene expression and PAL activity. At the same time, CCR activity only increased significantly in F. oxysporum-treated cells 24 h post elicitation. On the other hand, CAD activity measured for coniferyl alcohol formation was transiently decreased but a substrate-specific activation of CAD activity was observed in F. oxysporum-treated cells when using sinapyl alcohol as substrate. The accumulation of monolignol-derived products varied according to the elicitor used. B. cinerea or P. exigua-elicited cell cultures were characterised by a reinforcement of the cell wall by a deposit of 8-O-4′-linked non-condensed lignin structures and phenolic monomers, while at the same time no stimulation of 8-8′-linked lignan or 8-5′-linked phenylcoumaran lignan accumulation was observed. Additionally, elicitation of cell cultures with F. oxysporum extracts even triggered a strong incorporation of monolignols in the non condensed labile ether-linked lignin fraction concomitantly with a decrease in lignan and phenylcoumaran lignan accumulation. Several hypotheses are proposed to explain the putative role of these compounds in the defence response of flax cells against pathogens. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. C. Hano and M. Addi contributed equally to this work.     

Isolation and identification of an endophytic strain of Fusarium oxysporum producing podophyllotoxin from Juniperus recurva

Podophyllotoxin, a well-known naturally occurring aryltetralin lignan occurs in few plant species that is used as a precursor for the chemical synthesis of the anticancer drugs like etoposide, teniposide and etopophose phosphate. The availiability of this lignan is becoming increasingly limited because of the scarce occurance of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. This paper reports first time the production of podophyllotoxin by an endophytic fungus Fusarium oxysporum isolated from the medicinal plant Juniperus recurva. Further confirmation and quantification of podophyllotoxin was performed by HPLC, LC-MS, and LC-MS/MS.     

Increased lignan biosynthesis in the suspension cultures of Linum album by fungal extracts

Linum album accumulates anti-tumor podophyllotoxin (PTOX) and its related lignans, which were originally isolated from an endangered species Podophyllum. In the present study, we examined the effects of five fungal extracts on the production of lignans in L. album cell cultures. Fusarium graminearum extract induced the highest increase of PTOX [143 μg g⁻¹ dry weight (DW) of the L. album cell culture], while Rhizopus stolonifer extract enhanced the accumulation of lariciresinol up to 364 μg g⁻¹ DW, instead of PTOX. Typical elicitors, such as chitin, chitosan, or methyl jasmonate (MeJA), were shown to be less effective in lignan production in L. album cell cultures. These results verified the advantages of fungal extracts to increase lignan production in L. album cell culture, and suggested potential on-demand metabolic engineering of lignan biosynthesis using differential fungal extracts.     

Stereoselective Model Synthesis of the Optically Active Olivil Type of Lignan from D-Xylose

The olivil type of lignan, (2S,3R,4R)-4-benzyl-4- hydroxy-3-hydroxymethyl-2-(3,4-methylenedioxyphenyl)tetrahydrofuran, was stereoselectively synthesized from D-xylose.     

Insect feeding deterrent activity of lignans and related phenylpropanoids with a methylenedioxyphenyl (piperonyl) structure moiety

The antifeedant activity of a series of lignan lactones, hemiacetals, ethers, and alcohols derived from yatein and cubebin, together with structurally related phenylpropanoids and phenolics possessing a methylenedioxyphenyl (piperonyl) moiety, was tested against selected stored products pests: Sitophilus granarius L. (Coleoptera: Curculionidae), Tribolium confusum Duv. (Coleoptera: Tenebrionideae), and Trogoderma granarium Ev. (Coleoptera: Dermestridae). The relation between molecular structure and antifeedant activity was examined and implication of the piperonyl moiety is assessed. The compounds represent either natural substances isolated from plants (Libocedrus yateensis Guillaumin and Piper cubeba L.) or their structural analogues prepared by simple chemical transformations as well as compounds selected from commercially available sources. Natural lignan lactones with methoxy and/or methylenedioxy substituents showed significant activity that is strong enough to affect plant - insect interactions. Presence of polar substituents, especially hydroxy or glycosyl groups, often reduce the activity. Non-polar substituents, such as methoxy or methylenedioxy groups, enhance the activity not only in lignans but also in simple phenylpropanoids. The most active compound was synthetic piperonylbutoxide.     

Erimopyrone,a Lignan Derivative from the Liverwort Moerckia erimona

The new lignan derivative, erimopyrone, was isolated from the liverwort, Moerckia erimona. Its structure was established as [1R, 2S]-1(6-carboxy-2-oxo-2H-4-pyranyl)- 6,7-dihydroxy-1,2-dihydro-2,3-naphthalenedicarboxylic acid by spectroscopic methods.     

On the improvement of the podophyllotoxin production by phenylpropanoid precursor feeding to cell cultures of Podophyllum hexandrum Royle

In order to improve the production of the cytotoxic lignan podophyllotoxin, seven precursors from the phenylpropanoid-routing and one related compound were fed to cell suspension cultures derived from the rhizomes of Podophyllum hexandrum Royle. These cell cultures were able to convert only coniferin into podophyllotoxin, maximally a 12.8 fold increase in content was found. Permeabilization using isopropanol, in combination with coniferin as a substrate, did not result in an extra increase in podophyllotoxin accumulation. Concentrations of isopropanol exceeding 0.5% (v/v) were found to be rather toxic for suspension growth cells of P. hexandrum. When coniferin was fed in presence of such isopropanol concentrations, -glucosidase activity was still present, resulting in the formation of the aglucon coniferyl alcohol. In addition, podophyllotoxin was released into the medium under these permeabilization conditions. Entrapment of P. hexandrum cells in calcium-alginate as such or in combination with the feeding of biosynthetic precursors, did not improve the podophyllotoxin production. Cell-free medium from suspension cultures at later growth stages incubated with coniferin, resulted in the synthesis of the lignan pinoresinol.     

Knots in trees – A new rich source of lignans

Recent research in our group has revealed that knots, i.e. the branch bases inside tree stems, commonly contain 5–10% (w/w) of lignans. Norway spruce (Picea abies) knots contain as much as 6–24% of lignans, with 7-hydroxymatairesinol (HMR) as the predominant (70–85%) lignan. Some other spruce species also contain HMR as the main lignan, but some spruce species have also other dominating lignans. Most fir (Abies) species contain secoisolariciresinol and lariciresinol as the main lignans. Lignans occur also in knots of pines (Pinus spp.), although in lower amounts than in spruces and firs. Scots pine (Pinus silvestris) knots were found to contain 0.4–3% of lignans with nortrachelogenin as the main lignan. Lignans have been identified also in knots of some hardwoods, although flavonoids are more abundant in hardwoods. Knots are detrimental in the manufacture of pulp and paper and should preferably be removed before pulping. This is possible using a recently developed industrially applicable process called ChipSep. Recent research has also established novel synthetic routes to several lignans, such as matairesinol, secoisolariciresinol, lariciresinol and cyclolariciresinol, starting from hydroxymatairesinol by applying fairly straight-forward chemical transformations. We conclude that wood knots in certain spruce and fir species constitute the richest known source of lignans in nature. The lignans occur in knots in free form and are easily extracted by aqueous ethanol, or even by water. Not only HMR, but also other potentially valuable lignans, could be produced in a scale of hundreds of tons per year by extraction of knots separated from wood chips at pulp and paper mills.     

Lignans in plant cell and organ cultures: An overview

Lignans are found in a wide variety of plant species. The lignan podophyllotoxin is of special interest, since its derivatives like e.g. etopophos® are used in anticancer therapy. As chemical synthesis of podophyllotoxin is not yet economic, it still has to be isolated from wild growing Podophyllum species, some of which are considered to be endangered species. Therefore plant in vitro cultures may serve as alternative sources for podophyllotoxin and for other types of lignans as well. This review describes the establishment of plant cell and tissue cultures for lignan production and the experiments to improve product yields by changing the cultivation parameters, addition of elicitors and feeding of precursors. It also summarizes the use of plant cell and organ cultures to study the biosynthesis of lignans on enzymological level. Abbreviations: DOP – deoxypodophyllotoxin; LARI – lariciresinol; MATAI – matairesinol; 6MPTOX – 6-methoxypodophyllotoxin; PINO – pinoresinol; PTOX – podophyllotoxin; SECO – secoisolariciresinol     

Reactivity of bacterial and fungal laccases with lignin under alkaline conditions

The ability of Streptomyces ipomoea laccase to polymerize secoisolariciresinol lignan and technical lignins was assessed. The reactivity of S. ipomoea laccase was also compared to that of low redox fungal laccase from Melanocarpus albomyces using low molecular mass p-coumaric, ferulic and sinapic acid as well as natural (acetosyringone) and synthetic 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) mediators as substrates. Oxygen consumption measurement, MALDI-TOF MS and SEC were used to follow the enzymatic reactions at pH 7, 8, 9 and 10 at 30 °C and 50 °C. Polymerization of lignins and lignan by S. ipomoea laccase under alkaline reaction conditions was observed, and was enhanced in the presence of acetosyringone almost to the level obtained with M. albomyces laccase without mediator. Reactivities of the enzymes towards acetosyringone and TEMPO were similar, suggesting exploitation of the compounds and low redox laccase in lignin valorization under alkaline conditions. The results have scientific impact on basic research of laccases.     

Gadain,a lignan from jatropha gossypifolia

The isolation of a new lignan, gadain, from Jatropha gossypifolia is reported. The structure and stereochemistry of this compound have been determined from spectral analysis, partial synthesis from jatrophan (a lignan of known absolute configuration) and from its transformation reactions.     

The chain length of lignan macromolecule from flaxseed hulls is determined by the incorporation of coumaric acid glucosides and ferulic acid glucosides

Lignan macromolecule from flaxseed hulls is composed of secoisolariciresinol diglucoside (SDG) and herbacetin diglucoside (HDG) moieties ester-linked by 3-hydroxy-3-methylglutaric acid (HMGA), and of p-coumaric acid glucoside (CouAG) and ferulic acid glucoside (FeAG) moieties ester-linked directly to SDG. The linker molecule HMGA was found to account for 11% (w/w) of the lignan macromolecule. Based on the extinction coefficients and RP-HPLC data, it was determined that SDG contributes for 62.0% (w/w) to the lignan macromolecule, while CouAG, FeAG, and HDG contribute for 12.2, 9.0, and 5.7% (w/w), respectively.Analysis of fractions of lignan macromolecule showed that the higher the molecular mass, the higher the proportion of SDG was. An inverse relation between the molecular mass and the proportion (%) CouAG + FeAG was found. Together with the structural information of oligomers of lignan macromolecule obtained after partial saponification, it is hypothesized that the amount of CouAG + FeAG present during biosynthesis determines the chain length of lignan macromolecule.Furthermore, the chain length was estimated from a model describing lignan macromolecule based on structural and compositional data. The average chain length of the lignan macromolceule was calculated to be three SDG moieties with CouAG or FeAG at each of the terminal positions, with a variation between one and seven SDG moieties.     

An historical perspective on lignan biosynthesis: Monolignol,allylphenol and hydroxycinnamic acid coupling and downstream metabolism

This review describes discoveries from this laboratory on monolignol, allylphenol and hydroxycinnamic acid coupling, and downstream metabolic conversions, affording various lignan skeleta. Stereoselective 8-8′ coupling (dirigent protein-mediated) of coniferyl alcohol to afford (+)-pinoresinol is comprehensively discussed, as is our current mechanistic/kinetic understanding of the protein’s radical-radical binding, orientation and coupling properties, and insights gained for other coupling modes, e.g. affording (−)-pinoresinol. In a species dependent manner, (+)- or (−)-pinoresinols can also undergo enantiospecific reductions, catalyzed by various bifunctional pinoresinol-lariciresinol reductases (PLR), to afford lariciresinol and then secoisolariciresinol. With X-ray structures giving a molecular basis for differing PLR enantiospecificities, comparisons are made herein to the X-ray structure of the related enzyme, phenylcoumaran benzylic ether reductase, capable of 8-5′ linked lignan regiospecific reductions. Properties of the enantiospecific secoisolariciresinol dehydrogenase (also discovered in our laboratory and generating 8-8′ linked matairesinol) are summarized, as are both in situ hybridization and immunolocalization of lignan pathway mRNA/proteins in vascular tissues. This entire 8-8′ pathway thus overall affords secoisolariciresinol and matairesinol, viewed as cancer preventative agent precursors, as well as intermediates to cancer treating substances, such as podophyllotoxin derivatives. Another emphasis is placed on allylphenol/hydroxycinnamic acid coupling and associated downstream metabolism, e.g. affording the antiviral creosote bush lignan, nordihydroguaiaretic acid (NDGA), and the fern lignans, blechnic/brainic acids. Regiospecific 8-8′ allylphenol coupling is described, as is characterization of the first enantiospecific membrane-bound polyphenol oxidase, (+)-larreatricin hydroxylase, involved in NDGA formation. Specific [¹³C]-labeling also indicated that Blechnum lignans arise from stereoselective 8-2′ hydroxycinnamic acid coupling. Abbreviations: CD – circular dichroism; e.e. – enantiomeric excess; DP – dirigent protein; ESI-MS – electrospray ionization mass spectrometry; MALDI -TOF – matrix assisted laser desorption ionization-time of flight; MALLS – multiangle laser light scattering; PLR – pinoresinol lariciresinol reductase; SDH – secoisolariciresinol dehydrogenase. This revised version was published online in June 2006 with corrections to the Cover Date.     

Improved production of dibenzocyclooctadiene lignans in the elicited microshoot cultures of Schisandra chinensis (Chinese magnolia vine)

Dibenzocyclooctadiene lignans are a specific group of secondary metabolites that occur solely in Schisandra chinensis. The aim of the presented work was to boost the accumulation of lignans in the agitated microshoot cultures of S. chinensis, using different elicitation schemes. The experiments included testing of various concentrations and supplementation times of cadmium chloride (CdCl₂), chitosan (Ch), yeast extract (YeE), methyl jasmonate (MeJa), and permeabilizing agent—dimethylsulfoxide (DMSO). After 30 days, the microshoots were harvested and evaluated for growth parameters and lignan content by LC-DAD method. The analyses showed enhanced production of lignans in the elicited S. chinensis microshoots, whereas the respective media samples contained only trace amounts of the examined compounds (< 5 mg/l). Elicitation with CdCl₂ caused up to 2-fold increase in the total lignan content (max. ca. 730 mg/100 g DW after the addition of 1000 μM CdCl₂ on the tenth day). Experiments with chitosan resulted in up to 1.35-fold increase in lignan concentration (max. ca. 500 mg/100 g DW) after the supplementation with 50 mg/l on the first day and 200 mg/l on the tenth day. High improvement of lignan production was also recorded after YeE elicitation. After the elicitation with 5000 mg/l of YeE on the first day of the growth period, and with 1000 and 3000 mg/l on the 20th day, the lignan production increased to the same degree—about 1.8-fold. The supplementation with 1000 mg/l YeE on the 20th day of the growth cycle was chosen as the optimal elicitation scheme, for the microshoot cultures maintained in Plantform temporary immersion system—the total content of the estimated lignans was equal to 831.6 mg/100 g DW.

     

Effect of Various Elicitors on Lignan Biosynthesis in Callus Cultures of Linum austriacum

Effects of elicitors (mannan, -1,3-glucan, and ancymidol) on the activity of several key enzymes participating in lignan biosynthesis were studied in Linum austriacum L. cell cultures. The activities of L-phenylalanine ammonia-lyase, polyphenoloxydase, tyrosine ammonia-lyase, soluble phenoloxidase, and membrane-bound and soluble oxidases were assayed. The elicitors under study affected various steps in the metabolic pathway of lignan biosynthesis. Elevated enzyme activity accompanied an elicitor-enhanced synthesis of podophyllotoxins and peltatins.     

Stereochemical Difference in Secoisolariciresinol Formation between Cell-free Extracts from Petioles and from Ripening Seeds of Arctium lappa L.

Cell-free extracts from ripening seeds of Arctium lappa L. catalyzed the enantioselective formation of (-)-pinoresinol, (-)-lariciresinol and (-)-secoisolariciresinol from achiral coniferyl alcohol in the presence of NADPH and H₂O₂. The enantioselectivity of the lignan formation was opposite to that of the (+)-secoisolariciresinol formation catalyzed by cell-free extracts from petioles of the same plant species.     

Syntheses of ( + )-Phrymarolin II and Its Stereoisomers

Phrymarolin II, a unique naturally-occurring lignan having a 1,2-dioxygenated 3,7-dioxabicyclo[3.3.0]octane skeleton, was synthesized by the reaction of sesamol, in the presence of cadmium carbonate, with 1-acetoxy-2-chloro-6-(2′-methoxy-4′,5′-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octane. The latter was prepared through 13 steps starting from an aldol condensation of β-vinyl-γ-butyrolactone with 2-methoxy-4,5-methylenedioxybenzaldehyde. Three diastereomers of phrymarolin II were also obtained.