A selective de-O-methylation of guaiacyl lignans to corresponding catechol derivatives by 2-iodoxybenzoic acid (IBX). The role of the catechol moiety on the toxicity of lignans.

We report here the first selective de-O-methylation of a large panel of guaiacyl lignans to the corresponding catechol derivatives by using IBX as primary oxidant under green conditions (dimethyl carbonate-H(2)O solvent) through an in situ reduction procedure. The influence of the catechol moiety on the cytotoxicity and genotoxicity of new lignan derivatives has been investigated. The results obtained indicated that the presence of the catechol moiety sharply enhances the clastogenic potential (e.g. induction of chromosomal aberrations), the cytotoxicity and the modulation of cell cycle progression with respect to the parent compounds. Thus, despite the in vitro antioxidant activity usually described for catechol derivatives, our results show for the first time the generation of a clastogenic potential, highly indicative of a long-term genetic and cancer risk.

Structural investigation of biologically active phenolic compounds isolated from European tree species.

X-ray structures of two compounds isolated from wood knots of coniferous trees, namely dihydrokaempferol (3,5,8,13-tetrahydroxyflavanon) and lariciresinol (3,14-dimetoxy-7,10-epoxylignan-4,15,19-triol), are presented here. Diffraction data for the Dihydrokaempferol crystals were collected on a CAD4 diffractometer and on a synchrotron for the lariciresinol crystal. The investigated compounds inhibit lipid peroxidation and lariciresinol is additionally a good scavenger of superoxide radicals. The structural data presented in this work provide a useful basis for designing more active compounds with potential use as antioxidants.

Identification of lignans by liquid chromatography-electrospray ionization ion-trap mass spectrometry.

The fragmentation pattern of 30 compounds belonging to different classes of the lignan family was studied by liquid chromatography-electrospray ionization ion-trap mass spectrometry. On the basis of the observed fragmentation patterns, identification of different types of lignans was achieved. For example, dibenzylbutyrolactone lignans showed a characteristic fragmentation pathway by the loss of 44 Da (CO(2)) from the lactone moiety, whereas dibenzylbutanediols showed a loss of 48 Da by a combined loss of formaldehyde and water from the 1,4-butanediol moiety. Lignan glycosides readily lost the sugar residue to give the parent lignan as their primary product ion. In addition, several compound-specific fragmentations were observed and used for identification of individual compounds.A versatile method for analyses of lignans was developed using LC separation on a C8 column followed by fragmentation and detection of ions produced in the ion trap.

Complete assignments of the (1)H and (13)C chemical shifts and J(H,H) coupling constants in NMR spectra of D-glucopyranose and all D-glucopyranosyl-D-glucopyranosides.

Complete assignment of the (1)H and (13)C NMR spectra of all possible d-glucopyranosyl-d-glucopyranosides was performed and the (1)H chemical shifts and proton-proton coupling constants were refined by computational spectral analyses (using PERCH NMR software) until full agreement between the calculated and experimental spectra was achieved. To support the experimental results, the (1)H and (13)C chemical shifts and the spin-spin coupling constants between the non-hydroxyl protons of alpha- and beta-d-glucopyranose (1a and 1b) were calculated with density functional theory (DFT) methods at the B3LYP/pcJ-2//B3LYP/6-31G(d,p) level of theory. The effects of different glycosidic linkage types and positions on the glucose ring conformations and on the alpha/beta-ratio of the reducing end hydroxyl groups were investigated. Conformational analyses were also performed for anomerically pure forms of methyl d-glucopyranosides (13a and 13b) and fully protected derivatives such as 1,2,3,4,6-penta-O-acetyl-d-glucopyranoses (14a and 14b).

Quantification of a broad spectrum of lignans in cereals, oilseeds, and nuts.

Twenty-four plant lignans were analyzed by high-performance liquid chromatography-tandem mass spectrometry in bran extracts of 16 cereal species, in four nut species, and in two oilseed species (sesame seeds and linseeds). Eighteen of these were lignans previously unidentified in these species, and of these, 16 were identified in the analyzed samples. Four different extraction methods were applied as follows: alkaline extraction, mild acid extraction, a combination of alkaline and mild acid extraction, or accelerated solvent extraction. The extraction method was of great importance for the lignan yield. 7-Hydroxymatairesinol, which has not previously been detected in cereals because of destructive extraction methods, was the dominant lignan in wheat, triticale, oat, barley, millet, corn bran, and amaranth whole grain. Syringaresinol was the other dominant cereal lignan. Wheat and rye bran had the highest lignan content of all cereals; however, linseeds and sesame seeds were by far the most lignan-rich of the studied species.

The plant lignans matairesinol and secoisolariciresinol administered to Min mice do not protect against intestinal tumor formation.

The lignans matairesinol (MAT) and secoisolariciresinol (SECO) were fed to Min mice at 0.02% (w/w) in diet to study their effects on intestinal tumor development. The mean number (67 vs. 51, P=0.052) and size (1.4 vs. 1.2 mm, P=0.011) of tumors in the MAT group was elevated when compared with the control group. Tumor formation of the SECO group did not differ from the control group. Intake of MAT increased the level of both MAT and enterolactone in the plasma while SECO feeding increased SECO, enterodiol, and enterolactone (P=0.001). These results showed that MAT or SECO do not prevent intestinal carcinogenesis in Min mice and that MAT may have adverse effects.

Pinoresinol-lariciresinol reductases with different stereospecificity from Linum album and Linum usitatissimum.

Recently it was found that cell cultures and plants of Linum species contain lignans of various chemical structures. The stereochemistry of these compounds differ among species. Cell cultures of L. album accumulate (-)-podophyllotoxin together with pure (-)-secoisolariciresinol. The presence of both enantiomers of the precursor pinoresinol indicates that in L. album cell cultures the reactions from pinoresinol to secoisolariciresinol are the first steps determining enantiospecificity in biosynthesis of podophyllotoxin. Seeds of L. usitatissimum contain almost enantiomerically pure (+)-secoisolariciresinoldiglucosid derived from (+)-secoisolariciresinol. A cell culture of this species contains a mixture of both enantiomers of pinoresinol and pure (+)-secoisolariciresinol. In order to get more insight into the mechanism of (-)- and (+)-secoisolariciresinol biosynthesis, respectively, we isolated a cDNA encoding pinoresinol-lariciresinol reductase (PLR) from L. album. The heterologously expressed PLR-La1 converts only (+)-pinoresinol into (-)-secoisolariciresinol. In contrast, the heterologously expressed PLR from L. usitatissimum converts only (-)-pinoresinol to (+)-secoisolariciresinol confirming the results from others. Comparison of all available PLR protein sequences resulted in a few amino acids which may be responsible for the action of the PLRs with respect to the different enantioselectivity. A mutagenesis approach could not confirm this hypothesis. Aspects about the evolution of PLRs are discussed.

New lignan metabolites in rat urine.

Ten potential lignan metabolites were quantified in rat urine extracts using liquid chromatography-tandem mass spectrometry. The rats were orally administered with the plant lignans 7-hydroxymatairesinol, matairesinol, lariciresinol or secoisolariciresinol, or with the mammalian lignan enterolactone. The samples were enzymatically hydrolysed and solid-phase extracted before analysis. Of the analysed compounds, only trace amounts of 7-oxoenterolactone could be detected in the urine extracts before administration, but after administration of any of the lignans, the excretion of 7-oxoenterolactone increased and monodemethylated matairesinol and 4,4'-dihydroxyenterolactone could be detected. In addition, other novel lignan metabolites were detected, i.e., 7-oxomatairesinol, alpha-conidendrin, and alpha- and beta-conidendric acid.

Enterolactone inhibits the growth of 7,12-dimethylbenz(a)anthracene-induced mammary carcinomas in the rat.

The inverse association between a high enterolactone (ENL) concentration in both urine and serum, and the risk of breast cancer found in epidemiological studies suggests a chemopreventive action for ENL. However, no causal relationship has been established in clinical studies or in experimental models for breast cancer. In the present study, the potential chemopreventive action of p.o. administered ENL (1 or 10 mg/kg of body weight) was tested in 7,12-dimethylbenz(a)anthracene-induced mammary cancers of the rat. Rats were maintained on a standard open-formula chow diet. Daily p.o. administration of ENL at a dose of 10 mg/kg of body weight for 7 weeks significantly inhibited tumor growth. The growth-inhibitory effect of ENL was more pronounced on the new tumors, which developed during the treatment period, but ENL also inhibited the growth of those tumors established before the start of the lignan administration. The rat serum concentration of ENL, which illustrated a permanent positive effect on breast cancer growth, was 0.4 microM, which is >10-fold as compared with the serum concentrations found in the general human population. The effect of ENL was not restricted to any specific histological tumor type. ENL was demonstrated to act as a weak aromatase inhibitor in vitro and to reduce the relative uterine weight of the 7,12-dimethylbenz(a)anthracene-treated nonovariectomized rats. However, in a short-term assay ENL had no effect on the uterine growth of the intact or androstenedione-treated immature rats. Thus, the mechanism of the ENL action and its minimum or optimal daily dose remains to be clarified.

Synthesis of (-)-matairesinol, (-)-enterolactone, and (-)-enterodiol from the natural lignan hydroxymatairesinol.

[reaction: see text] We describe here a four-step semisynthetic method for the preparation of enantiomerically pure (-)-enterolactone starting from the readily available lignan hydroxymatairesinol from Norway spruce (Picea abies). Hydroxymatairesinol was first hydrogenated to matairesinol. Matairesinol was esterified to afford the matairesinyl 4,4'-bistriflate, which was deoxygenated by palladium-catalyzed reduction to 3,3'-dimethylenterolactone. Demethylation of 3,3'-dimethylenterolactone and reduction with LiAlH(4) yielded (-)-enterolactone and (-)-enterodiol, respectively.

Urinary excretion of lignans after administration of isolated plant lignans to rats: the effect of single dose and ten-day exposures.

The difference in urinary excretion of mammalian and plant lignans in rats was determined after oral administration of equivalent doses (25 mg/kg of body weight) of 7-hydroxymatairesinol (HMR), lariciresinol (LAR), matairesinol (MR), and secoisolariciresinol (SECO). Twenty-four hours-urine samples were collected after a single dose and after administration of one dose/day for 10 days. Eight lignans were analysed in urine extracts using a high-performance liquid chromatography-tandem mass spectrometry method showing good sensitivity and repeatability. After a single dose of HMR, LAR, MR, and SECO, the main metabolites were 7-hydroxyenterolactone (HENL), cyclolariciresinol (CLAR), enterolactone (ENL), and enterodiol (END), respectively, but after 10-day exposure ENL was the main metabolite of all the tested lignans, showing a considerably higher excretion than after a single dose. Metabolic transformations of plant lignans into each other could also be observed.

Structural determinants of plant lignans for the formation of enterolactone in vivo.

The quantity of mammalian lignans enterolactone (ENL) and enterodiol (END) and of plant lignans secoisolariciresinol (SECO) and 7-hydroxymatairesinol (HMR) excreted in a 24-h rat urine sample was measured after a single p.o. dose of an equivalent quantity of secoisolariciresinol diglycoside (SDG), secoisolariciresinol (SECO), matairesinol (MR), 7-hydroxymatairesinol (HMR) and ENL. Plant lignans (SECO and HMR) were partially absorbed as such. The aglycone form of SECO was more efficiently converted into mammalian lignans END and ENL than the glycosylated form, SDG. Of plant lignans, MR produced the highest quantities of ENL: the quantity was over twofold compared with HMR or SDG. The majority of the animals, which had been given SECO, excreted higher quantities of END than ENL into urine, but ENL was the main lignan metabolite after SDG. The highest quantities of ENL in urine were measured after the administration of ENL as such. The (-)SECO isolated from Araucaria angustifolia was converted into (-)ENL only. The administration of (-)SDG, which was shown to produce (+)SECO, resulted in excretion of (+)ENL only and (-)HMR was converted into (-)ENL only. This confirmed that the absolute configurations at C8 and C8' are not changed during the microbial metabolism. Whether the biological effects are enantiomer-specific, remains to be resolved.

Antioxidant activity of knotwood extractives and phenolic compounds of selected tree species.

The antioxidant potency and the radical scavenging capacity of superoxide and peroxyl radicals were assessed for 13 hydrophilic knotwood extracts of commercially important wood species, or fractions thereof, as well as for five pure wood-derived lignans and the flavonoid taxifolin. The chemical composition of the knotwood extracts was determined by gas chromatography combined with mass spectrometry. Most of the investigated wood species were rich in hydrophilic extractives (10-20% of the dry wood) with one or a few compounds dominating in each extract. All extracts had a high antioxidative potency and/or radical scavenging capacity as compared to the well-known antioxidants Trolox and butylated hydroxyanisole. The pure wood-derived lignans and taxifolin also had a high antioxidative potency and/or radical scavenging capacity. However, the antioxidant potency and/or radical scavenging capacity of several of the hydrophilic knotwood extracts were higher than that of the dominating compounds in pure form.

Fluorescent 7- and 8-methyl etheno derivatives of adenosine and 6-amino-9-ethylpurine: syntheses and fluorescence properties.

Two fluorescent adenosine derivatives (5 and 7) (Sch. 1) and two 6-amino-9-ethylpurine derivatives (6 and 8) (Sch. 1), were synthesised using 2-chloropropanal and 3-chloropropyne as reagents. The structures of the products were determined by spectroscopic and spectrometric methods (1H-, 13C- and 2D NMR, MS, UV and fluorescence spectrometry). Their fluorescence properties were determined and found to be similar to those of ethenoadenosine. Also, the stabilites of 5 and 7 in aqueous solutions were determined and found to be higher than that of the etheno derivative of adenosine.

Targeted allylation and propargylation of galactose-containing polysaccharides in water.

Galactose units of spruce galactoglucomannan (GGM), guar galactomannan (GM), and tamarind (galacto)xyloglucan (XG) were selectively allylated. Firstly aldehyde functionalities were formed at the C-6 position via enzymatic oxidation by galactose oxidase. The formed aldehydes were further derivatized by an indium mediated Barbier-Grignard type reaction, resulting in the formation of homoallylic alcohols. In addition to allylic halides, the same reaction procedure was also applicable for GGM, when using propargyl bromide as halide. All reaction steps were done in water, thus the polysaccharides were modified in a one-pot reaction. The formation of the allylated, or propargylated, product was identified by MALDI-TOF-MS. All polysaccharide products were isolated and further characterized by GC-MS or NMR spectroscopy. By this chemo-enzymatic process, we have demonstrated a novel method for derivatization of GGM and other galactose-containing polysaccharides. The derivatized polysaccharides are potential platforms for further functionalizations.

The antitumor lignan Nortrachelogenin sensitizes prostate cancer cells to TRAIL-induced cell death by inhibition of the Akt pathway and growth factor signaling.

Prostate cancer cells frequently develop resistance toward androgen-deprivation and chemotherapy. To identify new approaches to treat androgen-dependent prostate cancer, we have performed a structure-activity analysis of lignan polyphenols for cancer cell specific sensitization to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand that has ability to induce tumor-specific cell death. In this study, we report that the lignan nortrachelogenin (NTG) is the most efficient of the 27 tested lignan compounds in sensitizing prostate cancer cells to TRAIL-induced apoptosis. Importantly, pretreatment with NTG does not sensitize a non-malignant prostate cell line to TRAIL-induced cell death. The structural comparison of lignans reveals that the dibenzylbutyrolactone skeleton is required for the apoptosis-sensitizing activity, while substitutions at the aromatic rings do not seem to play a critical role in this lignan function. Our study also characterizes the cellular effects and molecular mechanisms involved in NTG anticancer activity. We previously reported that specific lignans inhibit the Akt survival-signaling pathway in concert with TRAIL sensitization. While NTG is also shown to be a effective inhibitor of Akt signaling, in this study we further demonstrate that NTG potently inhibits tyrosine kinase (RTK) activation in response to growth factors, such as insulin and insulin-like growth factor I (IGF-I). Our results identify NTG as a novel agent for prostate cancer therapy with ability to inhibit Akt membrane localization and activity as well as the activation of growth factor receptors (GFRs), thereby efficiently synergizing with TRAIL exposure.

Hydrophobication and characterisation of O-acetyl-galactoglucomannan for papermaking and barrier applications.

Norway spruce O-acetyl-galactoglucomannans (GGM) are water-soluble hemicelluloses that have potential to be produced in large scale as a side product of the mechanical pulping industry or by hot-water extraction of wood. Chemical modification is often needed to tailor such water-soluble polysaccharides into industrially valuable compounds. In this work, treatment of GGM with butyric and benzoic anhydride in pyridine/dimethylformamide rendered GGM derivatives, which were hydrophobic and partially soluble in organic solvents. The degree of substitution can be adjusted by varying the quantity of the reagent and reaction temperature. The dn/dc value for the benzoyl ester of GGM was determined in 0.05 M LiBr DMSO in order to obtain accurate molar mass analysis with SEC-MALLS-RI. Novel substances with adjusted hydrophobicity can thus be prepared on one step synthesis from natural hemicelluloses, which then showed a slight increase in the molar mass upon esterification.

Acetylation and characterization of spruce (Picea abies) galactoglucomannans.

Acetylated galactoglucomannans (GGMs) are the main hemicellulose type in most softwood species and can be utilized as, for example, bioactive polymers, hydrocolloids, papermaking chemicals, or coating polymers. Acetylation of spruce GGM using acetic anhydride with pyridine as catalyst under different conditions was conducted to obtain different degrees of acetylation on a laboratory scale, whereas, as a classic method, it can be potentially transferred to the industrial scale. The effects of the amount of catalyst and acetic anhydride, reaction time, temperature and pretreatment by acetic acid were investigated. A fully acetylated product was obtained by refluxing GGM for two hours. The structures of the acetylated GGMs were determined by SEC-MALLS/RI, (1)H and (13)C NMR and FTIR spectroscopy. NMR studies also indicated migration of acetyl groups from O-2 or O-3 to O-6 after a heating treatment in a water bath. The thermal stability of the products was investigated by DSC-TGA.

Metal-mediated allylation of enzymatically oxidized methyl α-D-galactopyranoside.

The C-6 unit of methyl α-D-galactopyranoside was selectively modified by combining enzymatic oxidation with an indium-mediated allylation reaction. The Barbier-Grignard type reaction, where a carbonyl group reacts with an allyl halide, proceeds in aqueous solution, even with water as the only solvent; thus carbohydrates can be modified without the need for drying or protection-deprotection steps. The corresponding homoallyl alcohols are produced in high yields of >90% in the reactions with allyl bromide and cinnamyl chloride. The main products were isolated and characterized by GC-MS and NMR spectroscopy.