Coniferyl alcohol hinders the growth of tobacco BY-2 cells and Nicotiana benthamiana seedlings.

MAIN CONCLUSION: Externally added coniferyl alcohol at high concentrations reduces the growth of Nicotiana cells and seedlings. Coniferyl alcohol is metabolized by BY-2 cells to several compounds. Coniferyl alcohol (CA) is a common monolignol and a building block of lignin. The toxicity of monolignol alcohols has been stated in the literature, but there are only few studies suggesting that this is true. We investigated the physiological effects of CA on living plant cells in more detail. Tobacco (Nicotiana tabacum) Bright yellow-2 cells (BY-2) and Nicotiana benthamiana seedlings both showed concentration-dependent growth retardation in response to 0.5-5 mM CA treatment. In some cases, CA addition caused cell death in BY-2 cultures, but this response was dependent on the growth stage of the cells. Based on LC-MS/MS analysis, BY-2 cells did not accumulate the externally supplemented CA, but metabolized it to ferulic acid, ferulic acid glycoside, coniferin, and to some other phenolic compounds. In addition to growth inhibition, CA caused the formation of a lignin-like compound detected by phloroglucinol staining in N. benthamiana roots and occasionally in BY-2 cells. To prevent this, we added potassium iodide (KI, at 5 mM) to overcome the peroxidase-mediated CA polymerization to lignin. KI had, however, toxic effects on its own: in N. benthamiana seedlings, it caused reduction in growth; in BY-2 cells, reduction in growth and cell viability. Surprisingly, CA restored the growth of KI-treated BY-2 cells and N. benthamiana seedlings. Our results suggest that CA at high concentrations is toxic to plant cells.

Oilseeds ameliorate metabolic parameters in male mice, while contained lignans inhibit 3T3-L1 adipocyte differentiation in vitro.

PURPOSE AND BACKGROUND: The focus was directed to the study of two of the most lignan-rich food sources: sesame and flaxseeds. Recent epidemiological and experimental evidences suggesting that these foods may improve metabolic functions underlying metabolic syndrome (MetS). METHODS: To characterize the effect of these oilseeds on metabolic functions, we conducted an experimental study aimed at preventing adiposity and metabolic imbalance in a mouse model of high-fat diet (HFD)-induced MetS. Statistical analysis was performed by two-way analysis of variance test followed by post hoc Bonferroni analysis. RESULTS: We studied the effect of the oilseeds sesame and flaxseed on metabolic parameters in mice on a HFD. When the HFD was integrated with 20% of sesame or flaxseed flours, the mice showed a decrease in body fat, already at day 15, from time 0. The size of the adipocytes was smaller in epididymal fat, liver steatosis was inhibited, and insulin sensitivity was higher in mice on the supplemented diets. The supplemented diets also resulted in a significant increase in the serum levels of the lignan metabolites enterodiol and enterolactone compared with the controls. The expression of genes associated with the inflammatory response, glucose metabolism, adipose metabolism and nuclear receptor were altered by the oilseed-supplemented diets. Some of the most abundant lignans in these oilseeds were studied in 3T3-L1 preadipocyte cells and were effective in inhibiting adipocyte differentiation at the minimal dose of 1 nM. CONCLUSIONS: The consumption of sesame and flaxseed may be beneficial to decrease metabolic parameters that are generally altered in MetS.

Pinaceae Pine Resins (Black Pine, Shore Pine, Rosin, and Baltic Amber) as Natural Dielectrics for Low Operating Voltage, Hysteresis-Free, Organic Field Effect Transistors.

Four pinaceae pine resins analyzed in this study: black pine, shore pine, Baltic amber, and rosin demonstrate excellent dielectric properties, outstanding film forming, and ease of processability from ethyl alcohol solutions. Their trap-free nature allows fabrication of virtually hysteresis-free organic field effect transistors operating in a low voltage window with excellent stability under bias stress. Such green constituents represent an excellent choice of materials for applications targeting biocompatibility and biodegradability of electronics and sensors, within the overall effort of sustainable electronics development and environmental friendliness.

Effect of dietary intervention on serum lignan levels in pregnant women - a controlled trial.

BACKGROUND: Mother's diet during pregnancy is important, since plant lignans and their metabolites, converted by the intestinal microflora to enterolignans, are proposed to possess multiple health benefits. Aim of our study was to investigate whether a dietary intervention affects lignan concentrations in the serum of pregnant women. METHODS: A controlled dietary intervention trial including 105 first-time pregnant women was conducted in three intervention and three control maternity health clinics. The intervention included individual counseling on diet and on physical activity, while the controls received conventional care. Blood samples were collected on gestation weeks 8-9 (baseline) and 36-37 (end of intervention). The serum levels of the plant lignans 7-hydroxymatairesinol, secoisolariciresinol, matairesinol, lariciresinol, cyclolariciresinol, and pinoresinol, and of the enterolignans 7-hydroxyenterolactone, enterodiol, and enterolactone, were measured using a validated method. RESULTS: The baseline levels of enterolactone, enterodiol and the sum of lignans were higher in the control group, whereas at the end of the trial their levels were higher in the intervention group. The adjusted mean differences between the baseline and end of the intervention for enterolactone and the total lignan intake were 1.6 ng/ml (p = 0.018, 95% CI 1.1-2.3) and 1.4 ng/mg (p = 0.08, 95% CI 1.0-1.9) higher in the intervention group than in the controls. Further adjustment for dietary components did not change these associations. CONCLUSION: The dietary intervention was successful in increasing the intake of lignan-rich food products, the fiber consumption and consequently the plasma levels of lignans in pregnant women. TRIAL REGISTRATION: ISRCTN21512277, http://www.isrctn.org.

Dietary lariciresinol attenuates mammary tumor growth and reduces blood vessel density in human MCF-7 breast cancer xenografts and carcinogen-induced mammary tumors in rats.

Lariciresinol is a dietary lignan that accounts for a significant portion of the total phytoestrogen intake from Western foods. Recent epidemiological studies suggest that high dietary intake of lignans and lariciresinol is associated with reduced breast cancer risk. However, no causal relationship between lariciresinol intake and breast cancer development has been established. In this study, we investigated for the first time the effects and possible mechanisms of action of lariciresinol on hormone responsive mammary cancer in vivo in dimethylbenz[a]anthracene induced mammary cancer in rats, and in human MCF-7 breast cancer xenografts in athymic mice. For tumor bearing rats, lariciresinol (3 or 15 mg/kg of body weight) or vehicle was administered p.o. daily for 9 weeks. For E2-maintained ovariectomized athymic mice bearing orthotopic MCF-7 tumors, control diet (AIN-93G) or lariciresinol containing diet (AIN-93G supplemented with 20 or 100 mg of lariciresinol/kg of diet) was administered for 5 weeks. In both models, lariciresinol administration inhibited the tumor growth and tumor angiogenesis. In MCF-7 cells, enterolactone significantly inhibited the E2-stimulated VEGF secretion. Moreover, in MCF-7 xenografts, lariciresinol administration enhanced tumor cell apoptosis and increased estrogen receptor beta expression. Lariciresinol and its further metabolites secoisolariciresinol, enterodiol and enterolactone were found in serum of both rats and athymic mice confirming a similar lignan metabolism pattern as in humans. These findings indicate conceivable importance of dietary lignan lariciresinol in inhibition of breast cancer development.

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.

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.

Determination of plant and enterolignans in human serum by high-performance liquid chromatography with tandem mass spectrometric detection.

An HPLC-MS/MS method was validated for the determination of the plant lignans 7-hydroxymatairesinol (HMR), matairesinol (Mat), secoisolariciresinol (Seco), lariciresinol (Lar), and cyclolariciresinol (CLar) and for the enterolignans 7-hydroxyenterolactone (HEL), enterodiol (ED), and enterolactone (EL) in human serum. The method included sample enzymatic hydrolysis, solid-phase extraction, and lignan analysis using a triple quadrupole mass spectrometer with electrospray ionisation in the multiple-reaction monitoring mode. The serum lignans were quantified using deuterated Mat or EL as internal standards. The method met the validation criteria for selectivity, intra- and inter-assay precision, and accuracy. The method was applied to ten serum samples collected from healthy individuals (five men and five women) consuming their habitual Finnish diet. All lignans except HMR and Seco were found in quantifiable amounts in the samples. All serums contained EL; the average concentration was 34 nM. In three individuals, the serum concentration of plant lignans was higher than that of enterolignans. Using the method, common dietary plant lignans and their major metabolites can be reliably quantified in human serum at low-nanomolar concentrations in a simple and rapid way.

Chemical characterization of high-molar-mass fractions in a Norway spruce knotwood ethanol extract.

The low-molar-mass (LMM) fraction, only, i.e., the GC-eluting compounds, which are mainly lignans, has been characterized in Norway spruce knotwood hydrophilic extracts previously. Of this fraction, many lignans and sesquilignans and all GC peaks supposedly representing dilignans remain unidentified. In this work, dilignans and the GC non-eluting compounds (the high-molar mass fractions, HMM) were characterized in a 7-hydroxymatairesinol-reduced knotwood ethanol extract of Norway spruce by using several fractionation and analytical techniques. A methyl tert-butyl ether (MTBE) insoluble fraction of the extract contained mainly HMM material, of which the main part was shown to consist of lignan oligomers. The oligolignans (with a molar mass up to approximately 3700 Da) seemed to be linked by 55' bonds, some of them containing one or two guaiacylglycerol ether units linked to the lignan by ßO4 or ß5 bonds. Several oligolignans were identified or tentatively identified. The MTBE soluble fraction, which accounted for the major part (81%) of the extract, contained mainly LMM material (lignans, sesqui- and dilignans). The part of the HMM material in the MTBE soluble fraction that was easily isolable (2%) seemed to contain polymers of fatty acids and alcohols, resin acids, and sterols.

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.

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.

Characterization of lignin extracted from birch wood by a modified hydrotropic process.

In this work an environmentally friendly hydrotropic process was used to extract lignin from industrial birch wood chips. Two hydrotropic treatments were performed, a conventional and a modified process. The lignins were characterized using FTIR, pyrolysis-gas chromatography-mass spectrometry (pyrolysis-GC-MS), (31)P and (1)H-(13)C HSQC NMR, and size exclusion chromatography (SEC). The chemical (carbohydrates, extractives, etc.) and elemental compositions of the lignins were also determined. The yields of both lignins were 16.1% (dry wood basis), and the obtained lignins had very low contents of non-lignin compounds. The treatments resulted in significant changes of the structure of the lignins, a decrease in aliphatic hydroxyls and an increase in phenolic ones. The lignin isolated by the modified treatment underwent more substantial change than the reference one. It is believed that the data presented will facilitate utilization of hydrotropic lignin and promote the adoption of the hydrotropic process in the pulp and biorefinery industry.

Interactions of a lignin-rich fraction from brewer's spent grain with gut microbiota in vitro.

Lignin is a constituent of plant cell walls and thus is classified as part of dietary fiber. However, little is known about the role of lignin in gastrointestinal fermentation. In this work, a lignin-rich fraction was prepared from brewer's spent grain and subjected to an in vitro colon model to study its potential bioconversions and interactions with fecal microbiota. No suppression of microbial conversion by the fraction was observed in the colon model, as measured as short-chain fatty acid production. Furthermore, no inhibition on the growth was observed when the fraction was incubated with strains of lactobacilli and bifidobacteria. In fact, the lignin-rich fraction enabled bifidobacteria to survive longer than with glucose. Several transiently appearing phenolic compounds, very likely originating from lignin, were observed during the fermentation. This would indicate that the gut microbiota was able to partially degrade lignin and metabolize the released compounds.

Content, composition, and stereochemical characterisation of lignans in berries and seeds.

In seed extracts of five oilseed species, in bran extracts of three cereal species, and in seed and/or whole berry extracts of 10 berry species, the concentrations of a large number of lignans and the enantiomeric composition of selected lignans were determined. In the case of sesame and hemp seeds, the lignan content and composition of the whole seeds was compared to that of the hulled seeds. The results showed that cloudberry seeds are the third most lignan-rich food source after linseeds and whole sesame seeds, and that most of the berry species analysed were more lignan-rich than the cereal brans. The lignans are concentrated in the hull of the oilseeds and in the seeds of the berries. In most samples, secoisolarici-, pino-, medio-, and syringaresinol were present as a mixture of two enantiomers.

Flaxseed ingestion alters ratio of enterolactone enantiomers in human serum.

Enterolactone (EL) is an enterolignan found in human subjects. In this pilot study, the enantiomeric ratios of serum EL were determined in serum from healthy adults during consumption of habitual diet, and after an 8-day supplementation with flaxseed (25 g/day). (-)EL dominated in all serum samples collected during habitual diet consumption. However, the ratio of (-)EL and (+)EL enantiomers differed markedly between individuals. Flaxseed ingestion increased significantly the proportion of (+)EL in all subjects. Moreover, a small but significant increase in serum (-)EL concentration was measured. After flaxseed ingestion, (-)EL concentrations correlated with those of (+)EL suggesting that the stereochemistry of the parent plant lignan in flaxseed is not a major determinant of EL formation in human subjects. Comparison of EL concentrations obtained with the validated chromatographic methods (HPLC-MS/MS, HPLC-CEAD, and GC-MS) and the time-resolved fluoroimmunoassay (TR-FIA) revealed that the immunoassay method underestimates human serum EL concentrations after the flaxseed ingestion.

Characterization of variation in the lignan content and composition of winter rye, spring wheat, and spring oat.

To characterize the range of variation in lignan content and composition caused by genotype and environment, seven dietary lignans, i.e., 7-hydroxymatairesinol, secoisolariciresinol, matairesinol, lariciresinol, pinoresinol, medioresinol, and syringaresinol, were analyzed by high-performance liquid chromatography-tandem mass spectrometry in whole-grain extracts of cereal samples collected at eight locations in Finland. In all, 28 winter rye, 73 spring wheat, and 55 spring oat samples were analyzed, representing 6, 9, and 5 cultivars, respectively. The total lignan content showed huge variations within the same cereal species: the range was 2500-6700 microg/100 g in the rye samples, 340-2270 microg/100 g in the wheat samples, and 820-2550 microg/100 g in the oat samples. The variations seemed to depend largely upon genetic differences. In rye, also environmental conditions affected the lignan content through grain size; smaller grains had significantly lower total lignan, syringaresinol, and lariciresinol content than larger grains. This study shows that varying cereal lignan concentrations reported in different studies may be, besides differences in analytical methods, largely dependent upon natural variations.

Occurrence of "mammalian" lignans in plant and water sources.

Enterolignans, also called "mammalian" lignans because they are formed in the intestine of mammals after ingestion of plant lignans, were identified for the first time in extracts of four tree species, i.e., in knot heartwood of the hardwood species Fagus sylvatica and in knot or stem heartwood of the softwood species Araucaria angustifolia, Picea smithiana, and Abies cilicia. They were also identified for the first time in grain extracts of cultivated plants, i.e., in 15 cereal species, in 3 nut species, and in sesame and linseeds. Furthermore, some plant lignans and enterolignans were identified in extracts of water from different sources, i.e., in sewage treatment plant influent and effluent and in humic water, and for the first time also in tap and seawater. They were present also in water processed through a water purification system (ultrapure water). As enterolignans seem to be abundant in the aquatic environment, the occurrence of enterolignans in plant sources is most likely due to uptake by the roots from the surrounding water. This uptake was also shown experimentally by treating wheat (Triticum aestivum ssp. vulgare) seeds with purified lignan-free water spiked with enterolactone (EL) during germination and growth. Both the remaining seeds and seedlings contained high EL levels, especially the roots. They also contained metabolites of EL, i.e., 7-hydroxy-EL and 7-oxo-EL.

Reactions of the natural lignan hydroxymatairesinol in basic and acidic nucleophilic media: formation and reactivity of a quinone methide intermediate.

The chemical properties and synthetic modifications of the natural lignan hydroxymatairesinol in basic and acidic nucleophilic media were studied. Hydroxymatairesinol presumably reacts via a quinone methide and a carbonium ion mechanism under basic and acidic conditions, respectively. In these conditions the benzylic hydroxyl group was displaced by nucleophiles yielding new 7-substituted butyrolactone lignans. Reactions in alcoholic basic solutions yielded the 7-alkoxy ethers diastereoselectively. Several previously known lignans as well as new lignans and lignan derivatives were synthesised. The transformations were monitored and the products identified by HPLC-MS and NMR.

A new lariciresinol-type butyrolactone lignan derived from hydroxymatairesinol and its identification in spruce wood.

When the natural lignan hydroxymatairesinol (1) was treated with an alkaline aqueous solution, it partially rearranged to isomeric forms of a lariciresinol-type butyrolactone lignan. The two major diastereomers formed (2 and 3) were isolated by column and medium-pressure chromatography, and their structures were elucidated by MS and NMR techniques. These previously unknown butyrolactone lignans were identified as naturally occurring in spruce knotwood by GC, GC-MS, and HPLC-ESI MS/MS analyses. The formation of isohydroxymatairesinol (2) and epi-isohydroxymatairesinol (3) from hydroxymatairesinol (1), and their detection in rat urine after administration of 1, is discussed.