Recovery of Bioactive Compounds from Hazelnuts and Walnuts Shells: Quantitative-Qualitative Analysis and Chromatographic Purification.

Hazelnut (HS) and walnut (WS) shells, an abundant by-product of the processing industries of these edible nuts, are traditionally considered as a low-value waste. However, they are a source of valuable compounds with an interesting chemical profile for the chemical and pharmaceutical sectors. In this study, the lipophilic and hydrophilic extracts present in HS and WS were quantified and identified, then the polar fractions were chromatographically separated, and their antioxidant capacity was studied. The experimental work includes the isolation of crude lipophilic and hydrophilic extracts by an accelerated extraction process, chromatographic analysis (gas chromatography-flame ionization (GC-FID), GC-mass spectroscopy (GC-MS), high-performance size-exclusion chromatography (HPSEC), thin-layer chromatography (TLC)), and quantification of the components. In addition, a thorough compositional characterization of the subgroups obtained by flash chromatography and their antioxidant capacity was carried out. The gravimetric concentrations showed different lipophilic/hydrophilic ratios (0.70 for HS and 0.23 for WS), indicating a higher proportion of polar compounds in WS than in HS. Moreover, the lipophilic extracts were principally composed of short-chain fatty acids (stearic, palmitic, and oleic acid), triglycerides, and sterols. The polar fractions were screened by thin-layer chromatography and then separated by flash chromatography, obtaining fractions free of fatty acids and sugar derivatives (97:3 in HS and 95:5 in WS), and mixtures richer in phenolic compounds and flavonoids such as guaiacyl derivatives, quercetin, pinobanksin, and catechin. The most polar fractions presented a higher antioxidant capacity than that of the crude extracts.

7-Hydroxymatairesinol improves body weight, fat and sugar metabolism in C57BJ/6 mice on a high-fat diet.

7-Hydroxymatairesinol (7-HMR) is a plant lignan abundant in various concentrations in plant foods. The objective of this study was to test HMRLignan™, a purified form of 7-HMR, and the corresponding Picea abies extract (total extract P. abies; TEP) as dietary supplements on a background of a high-fat diet (HFD)-induced metabolic syndrome in mice and in the 3T3-L1 adipogenesis model. Mice, 3 weeks old, were fed a HFD for 60 d. Subgroups were treated with 3 mg/kg body weight 7-HMR (HMRLignan™) or 10 mg/kg body weight TEP by oral administration. 7-HMR and TEP limited the increase in body weight (-11 and -13 %) and fat mass (-11 and -18 %) in the HFD-fed mice. Epididymal adipocytes were 19 and -12 % smaller and the liver was less steatotic (-62 and -65 %). Serum lipids decreased in TEP-treated mice (-11 % cholesterol, -23 % LDL and -15 % TAG) and sugar metabolism was ameliorated by both lignan preparations, as shown by a more than 70 % decrease in insulin secretion and insulin resistance. The expression of several metabolic genes was modulated by the HFD with an effect that was reversed by lignan. In 3T3-L1 cells, the 7-HMR metabolites enterolactone (ENL) and enterodiol (END) showed a 40 % inhibition of cell differentiation accompanied by the inhibited expression of the adipogenic genes PPARγ, C/EBPα and aP2. Furthermore, END and ENL caused a 10 % reduction in TAG uptake in HEPA 1-6 hepatoma cells. In conclusion, 7-HMR and TEP reduce metabolic imbalances typical of the metabolic syndrome and obesity in male mice, whereas their metabolites inhibit adipogenesis and lipid uptake in vitro.

Chemical Composition and Content of Lipophilic Seed Extractives of Some Abies and Picea Species.

The chemical content and composition of the lipophilic extracts from seeds of some fir species: Abies alba, A. cephalonica, A. concolor, and A. koreana, as well as of a few spruce species: Picea abies, P. orientalis, and P. pungens, were examined. The amount of lipophilic extractives is diverse among the tree species and it varies from 9.8% to 41% of seeds. The chemical characterization showed significant differences, not only in the content, but also in the composition of extractives. However, most of the identified compounds like resin alcohols, -aldehydes, and -acids, as well as fatty acids, were detected in the seed extracts of all the examined tree species. The dominating identified compound group was esterified fatty acids (2.5 - 55.4% w/w of dry extract), occurring mainly as tri- and diglycerides, as well as free acids. The main representatives of this group were linoleic and oleic acids. The resin acids, among which the main were abietic, neoabietic, dehydroabietic, and palustric acids, were also detected at high levels, from 1.8% to 16.9% of the dry seed extracts. Phytosterols, tocopherols, resin hydrocarbons, and resin esters, as well as fatty alcohols were also identified. The coniferous tree seeds, as a renewable natural material, could represent a prospective raw material for producing valuable chemicals.

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.

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.

Novel Lignan and stilbenoid mixture shows anticarcinogenic efficacy in preclinical PC-3M-luc2 prostate cancer model.

Prostate cancer is the most common cancer of men in the Western world, and novel approaches for prostate cancer risk reduction are needed. Plant-derived phenolic compounds attenuate prostate cancer growth in preclinical models by several mechanisms, which is in line with epidemiological findings suggesting that consumption of plant-based diets is associated with low risk of prostate cancer. The objective of this study was to assess the effects of a novel lignan-stilbenoid mixture in PC-3M-luc2 human prostate cancer cells in vitro and in orthotopic xenografts. Lignan and stilbenoid -rich extract was obtained from Scots pine (Pinus sylvestris) knots. Pine knot extract as well as stilbenoids (methyl pinosylvin and pinosylvin), and lignans (matairesinol and nortrachelogenin) present in pine knot extract showed antiproliferative and proapoptotic efficacy at ≥ 40 µM concentration in vitro. Furthermore, pine knot extract derived stilbenoids enhanced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis already at ≥ 10 µM concentrations. In orthotopic PC-3M-luc2 xenograft bearing immunocompromized mice, three-week peroral exposure to pine knot extract (52 mg of lignans and stilbenoids per kg of body weight) was well tolerated and showed anti-tumorigenic efficacy, demonstrated by multivariate analysis combining essential markers of tumor growth (i.e. tumor volume, vascularization, and cell proliferation). Methyl pinosylvin, pinosylvin, matairesinol, nortrachelogenin, as well as resveratrol, a metabolite of pinosylvin, were detected in serum at total concentration of 7-73 µM, confirming the bioavailability of pine knot extract derived lignans and stilbenoids. In summary, our data indicates that pine knot extract is a novel and cost-effective source of resveratrol, methyl pinosylvin and other bioactive lignans and stilbenoids. Pine knot extract shows anticarcinogenic efficacy in preclinical prostate cancer model, and our in vitro data suggests that compounds derived from the extract may have potential as novel chemosensitizers to TRAIL. These findings promote further research on health-related applications of wood biochemicals.

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.

The antimicrobial effects of wood-associated polyphenols on food pathogens and spoilage organisms.

The antimicrobial effects of the wood-associated polyphenolic compounds pinosylvin, pinosylvin monomethyl ether, astringin, piceatannol, isorhapontin, isorhapontigenin, cycloXMe, dHIMP, ArX, and ArXOH were assessed against both Gram-negative (Salmonella) and Gram-positive bacteria (Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus) and yeasts (Candida tropicalis, Saccharomyces cerevisiae). Particularly the stilbenes pinosylvin, its monomethyl ether and piceatannol demonstrated a clear antimicrobial activity, which in the case of pinosylvin was present also in food matrices like sauerkraut, gravlax and berry jam, but not in milk. The destabilization of the outer membrane of Gram-negative microorganisms, as well as interactions with the cell membrane, as indicated by the NPN uptake and LIVE/DEAD viability staining experiments, can be one of the specific mechanisms behind the antibacterial action. L. monocytogenes was particularly sensitive to pinosylvin, and this effect was also seen in L. monocytogenes internalized in intestinal Caco2 cells at non-cytotoxic pinosylvin concentrations. In general, the antimicrobial effects of pinosylvin were even more prominent than those of a related stilbene, resveratrol, well known for its various bioactivities. According to our results, pinosylvin could have potential as a natural disinfectant or biocide in some targeted applications.

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.

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.

Maternal flaxseed diet during pregnancy or lactation increases female rat offspring's susceptibility to carcinogen-induced mammary tumorigenesis.

Flaxseed contains several dietary components that have been linked to low breast cancer risk; i.e., n-3 polyunsaturated fatty acids (PUFAs), lignans and fiber, but it also contains detectable levels of cadmium, a heavy metal that activates the estrogen receptor (ER). Since estrogenic exposures early in life modify susceptibility to develop breast cancer, we wondered whether maternal dietary intake of 5% or 10% flaxseed during pregnancy or lactation (between postpartum days 5 and 25) might affect 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis in the rat offspring. Our data indicated that both in utero and postnatal 5% and 10% flaxseed exposures shortened mammary tumor latency, and 10% flaxseed exposure increased tumor multiplicity, compared to the controls. Further, when assessed in 8-week-old rats, in utero 10% flaxseed exposure increased lobular ER-alpha protein levels, and both in utero and postnatal flaxseed exposures dose-dependently reduced ER-beta protein levels in the terminal end buds (TEBs) lobules and ducts. Exposures to flaxseed did not alter the number of TEBs or affect cell proliferation within the epithelial structures. In a separate group of immature rats that were fed 5% defatted flaxseed diet (flaxseed source different than in the diets fed to pregnant or lactating rats) for 7 days, cadmium exposure through the diet was six-fold higher than allowed for humans by World Health Organization, and cadmium significantly accumulated in the liver and kidneys of the rats. It remains to be determined whether the increased mammary cancer in rats exposed to flaxseed through a maternal diet in utero or lactation was caused by cadmium present in flaxseed, and whether the reduced mammary ER-beta content was causally linked to increased mammary cancer risk among the offspring.

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.

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.