lignans and 2-3-bis(3--hydroxybenzyl)butane-1-4-diol

Lignans are diphenolic plant compounds with potential health modulating properties that are absorbed to the circulation and metabolized to the enterolignans enterodiol (END) and enterolactone (ENL) by gut microbiota. Epidemiological studies have inconsistently shown that a high lignan intake and circulating ENL are associated with reduced risk of breast-, prostate-, and colorectal cancer as well as cardiovascular disease and total and cause-specific mortality. Inconsistencies can be due to interpersonal variation of ENL formation or responses. The aim of this review is to identify and evaluate the impact of factors influencing variability in plasma concentrations of the main enterolignan, ENL. The main determinants of plasma ENL concentrations are intake of lignan and lignan-rich foods, composition and activity of intestinal microflora, antimicrobial use, nutrient intake, BMI, smoking, sex, and age. Composition and activity of the intestinal microbiota appear to be the most critical factor governing interpersonal variability in plasma ENL concentration followed by the use of antibiotics. Future studies with combined data from gut microbiota and metabolomics with food intake and life style data can be used to estimate the relative contribution of the different factors to ENL concentration in quantitative terms.

Topics: 4-Butyrolactone; Anti-Infective Agents; Body Mass Index; Female; Gastrointestinal Microbiome; Health Status; Humans; Lignans; Male; Nutrients

The analysis of experimental, clinical and epidemiological data on the phytoestrogen properties of the components of flaxseed Linum Usitatissimum L., especially lignans and products of their biotransformation in humans and animals enterodiol (END) and enterolactone (ENL) are presented. Flaxseed is the richest in the vegetable world source of lignans (up to 0,7-1,5% of dry weight of seed), among which prevails secoisolariciresinol diglycoside (SIR-DG). Plant lignans are characterized as natural phytoestrogen that reduce the risk of hormonedependent cancers of breast, uterus and prostate. Anticancerogenic activity of flaxseed lignans due to antioxudant effect END and ENL in the human body. The antioxidant activity of SIR, END, ENL and SIR-DG is higher than that of vitamin E and the antioxidant activity of SIR, END and ENL higher than SIR-DG. On the basis of evidence-based biomedical researches on various models in experimental carcinogenesis, on the tumor cells in vitro, in clinical trials in patients with hormone-dependent tumors, and, finally, in epidemiological studies have proved the anticarcinogenic activity of the components of the flaxseed and validity of recommendations for preventive and curative use in hormone-dependent tumors.

Topics: 4-Butyrolactone; Animals; Antioxidants; Flax; Humans; Lignans; Neoplasms, Experimental; Phytoestrogens

This review focuses on the possible role in human health of the consumption of lignan-rich foods. Most of the plant lignans in human foods are converted by the intestinal microflora in the upper part of the large bowel to enterolactone and enterodiol, called mammalian or enterolignans. The protective role of these compounds, particularly in chronic Western diseases, is discussed. Evidence suggests that fiber- and lignan-rich whole-grain cereals, beans, berries, nuts, and various seeds are the main protective foods. Many factors, in addition to diet, such as intestinal microflora, smoking, antibiotics, and obesity affect circulating lignan levels in the body. Lignan-rich diets may be beneficial, particularly if consumed for life. Experimental evidence in animals has shown clear anticarcinogenic effects of flaxseed or pure lignans in many types of cancer. Many epidemiological results are controversial, partly because the determinants of plasma enterolactone are very different in different countries. The source of the lignans seems to play a role because other factors in the food obviously participate in the protective effects. The results are promising, but much work is still needed in this area of medicine.

Topics: 4-Butyrolactone; Animals; Cardiovascular Diseases; Colorectal Neoplasms; Dietary Fiber; Edible Grain; Endometrial Neoplasms; Feeding Behavior; Female; Health Status; Humans; Isoflavones; Lignans; Male; Phytoestrogens; Plants, Edible; Prostatic Neoplasms; Seeds; Vegetables

The mammalian phytoestrogens enterodiol (END) and enterolactone (ENL) are produced in the colon by the action of bacteria on the plant precursors matairesinol (MAT), secoisolariciresinol (SECO), their glycosides, and other precursors in the diet. Both END and ENL have been shown to possess weakly estrogenic and antiestrogenic activities, and it has been suggested that the high production of these antiestrogenic mammalian lignans in the gut may serve to protect against breast cancer in women and prostate cancer in men. Various in vitro experiments suggested END and ENL significantly inhibited the growth of human colon tumor cells, and the E2-induced proliferation of MCF-7 breast cancer cells was inhibited by ENL. The protective effects of mammalian lignans may be due to their ability to compete with E2 for the type II estrogen receptor, to induce sex hormone binding globulin (SHBG), to inhibit placental aromatase, and to act as antioxidants. This review mainly deals with the chemistry, quantitative analysis, biological properties and health effects of END and ENL.

Topics: 4-Butyrolactone; Female; Humans; Lignans; Male; Tumor Cells, Cultured

Mammalian lignans are phytoestrogens with important bioactivities, and their concentrations in livestock milk may influence the health of consumers. This research aimed to establish a method to quantify multiple mammalian lignans in the biofluids of dairy sheep using ultra-HPLC-triple quadropole mass spectrometry with multiple-reaction monitoring. Secoisolariciresinol, 2-[(4-hydroxy-3-methoxyphenyl)methyl]-3-[(3-hydroxyphenyl)methyl]-1,4-butanediol, enterodiol (ED), enterolactone (EL), ED-sulfate (ED-S), and EL-sulfate (EL-S) were purified from the urine of flaxseed cake-fed dairy sheep. The structures of these lignans were identified by a combination of mass and nuclear magnetic resonance spectra. These purified lignans were used as standards to optimize their quantification conditions in urine, milk, and plasma of dairy sheep. On this basis, the lignan metabolites in biofluids were quantified. To improve analysis sensitivity, plasma and milk were pretreated with acetonitrile containing 1% formic acid and passed through a HybridSPE-PL 55261-U column (Supelco, Bellefonte, PA). The limit of quantification of the lignans ranged from 1.43 to 18.3 ng/mL in plasma, and from 1.01 to 18.7 ng/mL in milk. The linearity of the calibration curves ranged from their limit of quantification to at least 217 ng/mL in plasma, and 217 ng/mL in milk. Regression coefficient of the calibration curves were above 0.99 for secoisolariciresinol, 2-[(4-hydroxy-3-methoxyphenyl)methyl]-3-[(3-hydroxyphenyl)methyl]-1,4-butanediol, ED, EL, ED-S, and EL-S, indicating satisfactory relationships between the peak areas and concentrations in the quantification range. The relative concentrations of ED-glucuronide and EL-glucuronide (EL-G) in different biofluids were compared based on their chromatogram peak areas. The sheep plasma contained all forms of mammalian lignans (i.e., ED, EL, ED-S, EL-S, ED-glucuronide, and EL-G.); the urine contained ED, EL, ED-S, and EL-S; and the milk contained ED, EL, ED-S, EL-S, and EL-G. Milk-to-plasma concentration ratios of the mammalian lignans indicated that the free forms were more permeable than the sulfated conjugates. Mammalian lignans found in sheep plasma and milk may provide health benefits to the sheep and sheep-product consumers. The analytical method established in this work could be used to quantify mammalian lignans in livestock products.

Topics: 4-Butyrolactone; Animal Feed; Animals; Butylene Glycols; Flax; Glucosides; Lignans; Milk; Seeds; Sheep

This study aims at investigating the effect of an experimental period of intake of whole grain foods rich in lignans as part of an habitual diet on the plasma and urinary excretion of enterolignans, the biomarkers of lipid metabolism and the immunological and antioxidant status in a group of postmenopausal women with moderate serum cholesterol. A randomized double-blind crossover study was completed on 13 subjects in 12-weeks after protocol approval of an ethical committee. The subjects consumed whole grain foods high in lignans (30 g/d of breakfast cereals or biscuits, etc., 80 g/d of whole grain pasta) or refined grain foods for 4 weeks, separated by a 2-weeks wash-out period. A modest hypocholesterolemic effect (p < 0.05) of the whole grain diet was observed and the intake of whole grain products rich in lignans was also associated with an increase in urinary enterodiol excretion (p < 0.05).

Topics: Blood Pressure; Body Mass Index; Body Weight; Cholesterol; Cross-Over Studies; Diet; Double-Blind Method; Edible Grain; Female; Glutathione Peroxidase; Humans; Interleukin-1beta; Interleukin-6; Italy; Lignans; Middle Aged; Pilot Projects; Postmenopause; Superoxide Dismutase; Tumor Necrosis Factor-alpha

Enterolactone and enterodiol, mammalian lignans derived from dietary sources such as flaxseed, sesame seeds, kale, broccoli, and apricots, may impede tumor proliferation by inhibiting activation of nuclear factor kappa B (NFκB) and vascular endothelial growth factor (VEGF). We examined the associations between urinary enterolactone and enterodiol with prostatic tumor expression of NFκB, VEGF, and Ki67 among 147 patients with prostate cancer who participated in a presurgical trial of flaxseed supplementation (30 g/day) for ~30 days. Urinary enterolignans and tissue biomarkers were determined by high-performance liquid chromatography and immunohistochemistry, respectively. After supplementation, we observed significant correlations between intakes of plant lignan and urinary concentrations of total enterolignans (ρ=0.677, P<.0001), enterolactone (ρ=0.676, P<.0001), and enterodiol (ρ=0.628, P<.0001). Importantly, we observed that total urinary enterolignans and enterolactone were significantly and inversely correlated with Ki67 in the tumor tissue (ρ=-0.217, P=.011, and ρ=-0.230, P=.007, respectively), and a near-significant inverse association was observed for enterodiol (ρ=-0.159, P=.064). An inverse association was observed between enterolactone and VEGF (ρ=-0.143, P=.141), although this did not reach statistical significance. We did not observe an association between enterolignans and NFκB. In conclusion, flaxseed-derived enterolignans may hinder cancer cell proliferation via VEGF-associated pathways.

Topics: 4-Butyrolactone; Aged; Antineoplastic Agents, Phytogenic; Biomarkers; Cell Proliferation; Dietary Supplements; Flax; Humans; Ki-67 Antigen; Lignans; Male; Middle Aged; Phytotherapy; Plant Extracts; Prostatic Neoplasms; Vascular Endothelial Growth Factor A

The mammalian lignans enterolactone and enterodiol, which are produced by the microflora in the colon of humans and animals from precursors in foods, have been suggested to have potential anticancer effects. This study determined the production of mammalian lignans from precursors in food bars containing 25 g unground whole flaxseed (FB), sesame seed (SB), or their combination (FSB; 12.5 g each). In a randomized crossover study, healthy postmenopausal women supplemented their diets with the bars for 4 wk each separated by 4-wk washout periods, and urinary mammalian lignan excretion was measured at baseline and after 4 wk as a marker of mammalian lignan production. Results showed an increase with all treatments (65.1-81.0 mumol/day; P < 0.0001), which did not differ among treatments. Lignan excretion with the whole flaxseed was similar to results of other studies using ground flaxseed. An unidentified lignan metabolite was detected after consumption of SB and FSB but not of FB. Thus, we demonstrated for the first time that 1) precursors from unground whole flaxseed and sesame seed are converted by the bacterial flora in the colon to mammalian lignans and 2) sesame seed, alone and in combination with flaxseed, produces mammalian lignans equivalent to those obtained from flaxseed alone.

Topics: 4-Butyrolactone; Administration, Oral; Aged; Cross-Over Studies; Female; Flax; Humans; Lignans; Phytoestrogens; Postmenopause; Seeds; Sesamum

Flaxseed is one of the richest sources of lignans and is increasingly used in food products or as a supplement. Plant lignans can be converted by intestinal bacteria into the so-called enterolignans, enterodiol and enterolactone. For a proper evaluation of potential health effects of enterolignans, information on their bioavailability is essential. The aim of this study was to investigate whether crushing and milling of flaxseed enhances the bioavailability of enterolignans in plasma. In a randomized, crossover study, 12 healthy subjects supplemented their diet with 0.3 g whole, crushed, or ground flaxseed/(kg body weight . d). Each subject consumed flaxseed for 10 successive days separated by 11-d run-in/wash-out periods, in which the subjects consumed a diet poor in lignans. Blood samples were collected at the end of each run-in/wash-out period, and at the end of each supplement period. Plasma enterodiol and enterolactone were measured using LC-MS-MS. The mean relative bioavailability of enterolignans from whole compared with ground flaxseed was 28% (P < or = 0.01), whereas that of crushed compared with ground flaxseed was 43% (P < or = 0.01). Crushing and milling of flaxseed substantially improve the bioavailability of the enterolignans.

Topics: 4-Butyrolactone; Adolescent; Adult; Biological Availability; Cross-Over Studies; Diet; Dietary Supplements; Female; Flax; Food Handling; Humans; Lignans; Male; Middle Aged; Seeds

The mammalian lignans enterolactone and enterodiol are produced in the colon by the action of bacteria on the plant precursor secoisolariciresinol diglycoside, which is found in high concentrations in flaxseed.. Two experiments were conducted to determine 1) whether there is a dose response in urinary lignan excretion with increasing flaxseed intake, 2) whether flaxseed processing affects lignan excretion, 3) peak plasma lignan concentrations, and 4) plasma lignan concentrations after chronic supplementation.. Nine healthy young women supplemented their diets with 5, 15, or 25 g raw or 25 g processed (muffin or bread) flaxseed for 7 d during the follicular phase of their menstrual cycles. Twenty-four-hour urine samples were collected at baseline and on the final day of supplementation. As an adjunct to the 25-g-flaxseed arm, subjects consumed the supplement for an additional day and blood and urine samples were collected at specific intervals. All blood and urine samples were analyzed for enterolactone and enterodiol by gas chromatography-mass spectroscopy.. A dose-dependent urinary lignan response to raw flaxseed was observed (r = 0.72, P < 0.001). The processing of flaxseed as a muffin or bread did not affect the quantity of lignan excretion. Plasma lignan concentrations were greater (P < or = 0.05) than baseline by 9 h after flaxseed ingestion (29.35+/-3.69 and 51.75+/-7.49 nmol/L, respectively). The total plasma area under the curve was higher on the eighth than on the first day (1840.15+/-343.02 and 1027.15+/-95.71 nmol x h/L, respectively).. Mammalian lignan production from flaxseed precursors is dependent on time and dose but not on processing.

Topics: 4-Butyrolactone; Administration, Oral; Adult; Body Mass Index; Cross-Over Studies; Dietary Fiber; Dose-Response Relationship, Drug; Double-Blind Method; Female; Flax; Follicular Phase; Gas Chromatography-Mass Spectrometry; Humans; Lignans; Seeds; Time Factors

Enterolactone (ENL) and enterodiol (END) are found in high concentrations in human body fluids after ingestion of flaxseed and whole-grain products. Although much interest is presently focused on these mammalian lignans because of their putative beneficial health effects, little is known about their metabolic fate in humans. We have now identified nine novel metabolites of ENL and END in the urine of female and male humans ingesting flaxseed for five days. The chemical structures of six ENL metabolites and of three END metabolites were elucidated by GC/MS analysis and comparison with authentic reference compounds obtained by chemical synthesis. The six identified metabolites of ENL were the products of monohydroxylation at the para-position and at both ortho-positions of the parent hydroxy group of either aromatic ring. Likewise, the three END metabolites were formed through aromatic monohydroxylation at the para- and ortho-positions. The biological significance of these metabolites remains to be established.

Topics: 4-Butyrolactone; Female; Flax; Gas Chromatography-Mass Spectrometry; Humans; Lignans; Male; Molecular Structure; Seeds

The aim of this study was to test the hypothesis that increased dietary intake of phytoestrogens reduces the health impact of the menopause. To test this hypothesis, a double-blind, randomized, entry-exit, cross-over study was conducted to assess the effects of three dietary manipulations--soy and linseed diets (high in phytoestrogens) and a wheat diet (low in phytoestrogens). Postmenopausal women were recruited and randomly assigned to one of the three dietary regimens. Urinary phytoestrogen concentrations, hot flush rate, vaginal smears, bone mineral density and bone mineral content were assessed for two 12-week periods. Comparative analysis showed no significant differences, but, when analyzed separately, groups consuming high phytoestrogen diets had between 10 and 30 times higher urinary excretion of phytoestrogens compared to those consuming the low phytoestrogen diet (p < 0.01). Study participants consuming soy, linseed and wheat diets had a 22% (not significant, n.s.), 41% (p < 0.009) and 51% (p < 0.001) reduction in hot flush rate; a 103% (p < 0.04), 5.5% (n.s.) and 11% (n.s.) increase in vaginal cytology maturation index; and a 5.2% (p < 0.04), 5.2% (n.s.) and 3.8% (n.s.) increase in bone mineral content, respectively. No changes were detected in bone mineral density. The differential effects of high phytoestrogen dietary manipulations on outcomes may represent tissue-specific responses to isoflavones and lignans contained in soy and linseed, respectively. Whilst health outcome measures were not significantly different between groups, the data obtained from separate analysis suggest that phytoestrogens in soy and linseed may be of use in ameliorating some of the symptoms of menopause. Furthermore, the significant decrease in hot flush rate in the wheat group cannot be attributable to phytoestrogens measured in this study. Due to subject variability, larger studies are still needed to evaluate population benefit.

Topics: 4-Butyrolactone; Aged; Bone Density; Diet; Double-Blind Method; Estrogens, Non-Steroidal; Female; Genistein; Glycine max; Hot Flashes; Humans; Isoflavones; Lignans; Linseed Oil; Middle Aged; Phytoestrogens; Plant Preparations; Postmenopause; Triticum

Urinary lignan and isoflavonoid excretion were examined in 11 men and 9 women consuming four nine-day controlled experimental diets: basal (vegetable free), carotenoid vegetable (carrot and spinach), cruciferous vegetable (broccoli and cauliflower), and soy (tofu and textured vegetable protein product). Three-day urine collections (Days 7-9) were analyzed for lignans and isoflavonoids with use of isotope-dilution gas chromatography-mass spectrometry. Urinary excretion of the lignans enterodiol and enterolactone was higher during the carotenoid and cruciferous vegetable diets than during the basal diet (p = 0.0001), suggesting that these vegetables may provide a source of mammalian lignan precursors. Urinary excretion of the isoflavonoids equol, O-desmethylangolensin, daidzein, and genistein was higher when subjects consumed soy diets than when they consumed the other test diets (p < 0.02). Gender differences in lignan excretion were observed. Men excreted more enterolactone (p = 0.006) and less enterodiol (p = 0.013) than women, implying a gender difference in colonic bacterial metabolism of lignans. There was no effect of gender on isoflavonoid excretion.

Topics: 4-Butyrolactone; Adult; Chromans; Colon; Cross-Over Studies; Diet; Equol; Female; Gas Chromatography-Mass Spectrometry; Genistein; Glycine max; Humans; Isoflavones; Lignans; Male; Sex Characteristics; Vegetables

To compare the effects of consumption of fermented and unfermented soy products on excretion of urinary isoflavonoid phytoestrogens and lignans in healthy men.. A randomized, crossover trial consisting of two 9-day feeding periods following 5 days of baseline data collection.. Healthy men, aged 20 to 40 years, were recruited from the University of Minnesota Twin Cities community. Of the 22 subjects who began the study, 17 completed all feeding periods.. Fermented soy product (112 g tempeh) or unfermented soy (125 g soybean pieces) was consumed during each controlled feeding period.. Urine samples collected while subjects consumed their habitual diets and on the last 3 days of each feeding period were analyzed for isoflavonoid and lignan content by isotope dilution gas chromatography-mass spectrometry.. Comparisons of isoflavonoid and lignan excretion were analyzed using the general linear model procedure. Orthogonal contrasts were used to determine treatment differences of interest.. Urinary excretion of isoflavonoids (equol, O-desmethylangolensin [O-DMA], daidzein, genistein) was higher and excretion of lignans (enterodiol, enterolactone) was lower when subjects consumed soy-supplemented diets than when they consumed their habitual diets (P < .05). Urinary isoflavonoid excretion and lignan excretion were similar when subjects consumed tempeh and soybean pieces diets; however, recovery of daidzein and genistein was significantly higher when subjects consumed the tempeh diet than when they consumed the soybean pieces diet (P < .002). When fed soy, 5 of 17 subjects excreted high amounts of equol. These five subjects tended to excrete less O-DMA and daidzein than the 12 subjects who excreted low amounts of equol (P < .06).. Fermentation of soy decreased the isoflavone content of the product fed but increased the urinary isoflavonoid recovery. This finding suggests that fermentation increases availability of isoflavones in soy.

Topics: 4-Butyrolactone; Adult; Chromans; Cross-Over Studies; Defecation; Diet; Equol; Estrogens; Estrogens, Non-Steroidal; Fermentation; Food Handling; Genistein; Glycine max; Humans; Isoflavones; Lignans; Male; Phytoestrogens; Plant Preparations; Taste

To compare the effect of vegetable, fruit, and legume consumption on urinary isoflavonoid phytoestrogen and lignan excretion.. After 4 days of data collection, during which subjects consumed their habitual diets, subjects were randomly placed on four 9-day controlled experimental diets with each subject receiving each diet in a random order.. Seven men and three women, aged 20 to 35 years, were recruited from the University of Minnesota Twin Cities community.. All subjects consumed four experimental diets in an assigned random order: a controlled basal diet, a legume/allium diet (containing garbanzo beans, garlic, and onions), and diets low or high in vegetables and fruits (containing apples, pears, potatoes, and carrots).. Urine samples that were collected while subjects consumed their habitual diets and during the last 3 days of each feeding period were analyzed for isoflavonoid and lignan content using isotope dilution gas chromatography-mass spectrometry.. The effect of vegetable and fruit intake on urinary isoflavonoid and lignan excretion was analyzed using the general linear model procedure. Post hoc comparisons were made using Duncan's multiple range test.. Subjects excreted more of the lignan enterodiol on the high vegetable/fruit diet compared with the basal and legume/allium diets (P = .03); more of the isoflavonoids O-desmethylangolensin (O-DMA), genistein, and sum of isoflavonoids on the legume/allium diet compared with the other controlled diets (P < .05); and more of the isoflavan equol on the basal and legume/allium diets compared with the high vegetable/fruit diet (P < .01). Subjects who excreted higher levels of equol on the basal and legume/allium diets also consumed more of the milk-based pudding provided as part of the controlled diets.. Urinary lignan and isoflavonoid excretion changed in response to alterations in vegetable, fruit, and legume intake under controlled dietary conditions.

Topics: Adult; Chromans; Cross-Over Studies; Diet; Dietary Carbohydrates; Dietary Fats; Dietary Fiber; Energy Intake; Equol; Estrogens, Non-Steroidal; Fabaceae; Female; Flavonoids; Fruit; Genistein; Humans; Isoflavones; Lignans; Male; Phytoestrogens; Plant Preparations; Plants, Medicinal; Vegetables

Lignans are a group of phytochemicals shown to have weakly estrogenic and antiestrogenic properties. Two specific lignans, enterodiol and enterolactone, are absorbed after formation in the intestinal tract from plant precursors particularly abundant in fiber-rich food and are excreted in the urine. We evaluated the effect of the ingestion of flax seed powder, known to produce high concentrations of urinary lignans, on the menstrual cycle in 18 normally cycling women, using a balanced randomized cross-over design. Each subject consumed her usual omnivorous, low fiber (control) diet for 3 cycles and her usual diet supplemented with flax seed for another 3 cycles. The second and third flax cycles were compared to the second and third control cycles. Three anovulatory cycles occurred during the 36 control cycles, compared to none during the 36 flax seed cycles. Compared to the ovulatory control cycles, the ovulatory flax cycles were consistently associated with longer luteal phase (LP) lengths (mean +/- SEM, 12.6 +/- 0.4 vs. 11.4 +/- 0.4 days; P = 0.002). There were no significant differences between flax and control cycles for concentrations of either estradiol or estrone during the early follicular phase, midfollicular phase, or LP. Although flax seed ingestion had no significant effect on LP progesterone concentrations, the LP progesterone/estradiol ratios were significantly higher during the flax cycles. Midfollicular phase testosterone concentrations were slightly higher during flax cycles. Flax seed ingestion had no effect on early follicular phase concentrations of DHEA-S, PRL, or sex hormone-binding globulin. Our data suggest a significant specific role for lignans in the relationship between diet and sex steroid action, and possibly between diet and the risk of breast and other hormonally dependent cancers.

Topics: 4-Butyrolactone; Adult; Diet; Estrogens; Female; Hormones; Humans; Lignans; Luteal Phase; Menstrual Cycle; Osmolar Concentration; Plants, Edible

Colorectal cancer (CRC), especially metastatic (mCRC) form, becomes a major reason behind cancer morbidity worldwide, whereas the treatment strategy is not optimum. Several novel targets are under investigation for mCRC including the autophagy pathway. Natural compounds including dietary lignans are sparsely reported as autophagy modulators. Nonetheless, the interaction between dietary lignans and core autophagy complexes are yet to be characterised. We aimed to describe the interaction between the dietary lignans from flaxseed (Linum usitatissimum) and sesame seeds (Sesamum indicum) along with the enterolignans (enterodiol and enterolactone) and the UNC-51-like kinase 1 and 2 (ULK1/2), important kinases required for the autophagy. A range of in-silico technologies viz. molecular docking, drug likeness, and ADME/T was employed to select the best fit modulator and/or inhibitor of the target kinases from the list of selected lignans. Drug likeness and ADME/T studied further selected the best-suited lignans as potential autophagy inhibitor. Molecular dynamic simulation (MDS) analyses were used to validate the molecular docking results. Binding free energies of the protein-ligand interactions by MM-PBSA method further confirmed best-selected lignans as ULK1 and/or ULK2 inhibitor. In conclusion, three dietary lignans pinoresinol, medioresinol, and lariciresinol successfully identified as dual ULK1/2 inhibitor/modifier, whereas enterodiol emerged as a selective ULK2 inhibitor/modifier.

Topics: Autophagy; Autophagy-Related Protein-1 Homolog; Colorectal Neoplasms; Humans; Lignans; Molecular Docking Simulation

Phytoestrogens may have potential effects on hormone-related cancers (HRC) and cancer biomarkers, but the findings have been inconsistent so far. Participants from the National Health and Nutrition Examination Survey 1999-2010 with information on the levels of urinary phytoestrogens, serum cancer biomarkers and cancer history were included. Sampling-weighted logistic regression models examined the association between urinary phytoestrogens concentrations (creatinine-standardised and log-transformed) and HRC, followed by stratified analyses by race/ethnicity, age and menopausal status for different gender. Correlation analyses between phytoestrogens and cancer biomarkers were performed. Of the total 8844 participants, there were 373 with HRC. We observed total isoflavone and enterodiol excretion were positively associated with HRC, especially in non-Hispanic white female subpopulations (

Topics: Biomarkers, Tumor; Humans; Isoflavones; Lignans; Male; Nutrition Surveys; Phytoestrogens; Prostate-Specific Antigen; Prostatic Neoplasms

Secoisolariciresinol diglucoside (SDG) is the principal lignan of flaxseed and precursor of its aglycone, secoisolariciresinol (SECO), and the mammalian lignans enterolactone (EL) and enterodiol (ED), the putative bioactive forms of oral administration of SDG. SDG is present in the seed hull as an ester-linked polymer. Although extraction and purification of SDG monomer is costly, the use of naturally occurring SDG in polymer form may offer a more economical approach for delivery of this precursor. The extent of SDG release from the polymer and subsequent bioavailability of SDG metabolites are unknown. To understand the relative bioavailability of SDG polymer, this study examined the comparative bioavailability of enriched SDG and SDG polymer in rats after a single oral SDG equivalent dose (40 mg/kg). A validated LC-MS/MS method quantified SDG and its metabolites in rat plasma following serial blood collections. SDG remained undetectable in rat plasma samples. Unconjugated SECO was detected in plasma after 0.25 h. Unconjugated ED was observed after 8 h (3.4 ± 3.3 ng/mL) and 12 h (6.2 ± 3.3 ng/mL) for enriched SDG and SDG polymer, respectively. Total (conjugated and unconjugated) ED and EL resulting from enriched SDG and SDG polymer reached similar maximal concentrations between 11 and 12 h and demonstrated similar total body exposures (AUC values). These data suggest a similar pharmacokinetic profile between the enriched and polymer form of SDG, providing support for the use of SDG polymer as a more economical precursor for SECO, ED, and EL in applications of chronic disease management.

Topics: 4-Butyrolactone; Animals; Biological Availability; Butylene Glycols; Female; Flax; Glucosides; Lignans; Molecular Structure; Polymers; Rats; Rats, Wistar; Seeds

Topics: Antioxidants; Atmosphere; Carbon Dioxide; Chromatography, Liquid; Cicer; Digestion; Edible Grain; Flavonoids; Lignans; Mass Spectrometry; Nitrogen; Phenols; Plant Proteins

Current evidence on the relationship of phytoestrogens with sleep is limited and contradictory. In particular, studies on individual phytoestrogens and sleep have not been reported. Thus, this study aimed to appraise the associations of individual phytoestrogens with sleep disorders and sleep duration. This cross-sectional study comprising 4830 adults utilized data from the National Health and Nutrition Examination Survey 2005-2010. Phytoestrogens were tested in urine specimens. Sleep disorders and sleep duration were based on a self-reported doctor's diagnosis and usual sleep duration. The main analyses utilized logistic and multinomial logistic regression models and a restricted cubic spline. In the fully adjusted model, compared with tertile 1 (lowest), the odds ratios (95% confidence intervals (CIs)) of sleep disorders for the highest tertile of urinary concentrations of enterolactone, enterodiol, and O-desmethylangolensin were 0.64 (0.41-1.00), 1.54 (1.07-2.21), and 1.89 (1.26-2.85), respectively. Linear inverse, approximatively linear positive, and inverted L-shaped concentration-response relationships were found between enterolactone, enterodiol, and O-desmethylangolensin and sleep disorders, respectively. Compared with normal sleep (7-8 h/night), the relative risk ratio (RRR) (95% CI) of very short sleep for enterolactone was 0.56 (0.36-0.86), and the RRR (95% CI) of long sleep risk for genistein was 0.62 (0.39-0.99). Furthermore, negative associations of genistein with sleep disorders and enterolactone with long sleep risk, as well as positive associations of enterodiol with both long and very short sleep, were observed in the stratified analysis by age or gender. Finally, a notable finding was that urinary O-desmethylangolensin concentration was positively related to sleep disorders in both females aged 40-59 years and non-Hispanic Whites but inversely associated with sleep disorders in both females aged 60 years or over and other Hispanics. Our findings suggested that enterolactone and genistein might be beneficial for preventing sleep disorders or non-normal sleep duration among adults, and enterodiol might be adverse toward this goal. However, the association of O-desmethylangolensin with sleep disorders might be discrepant in different races and females of different ages.

Topics: 4-Butyrolactone; Adult; Age Factors; Aged; Biomarkers; Cross-Sectional Studies; Female; Humans; Isoflavones; Lignans; Male; Middle Aged; Phytoestrogens; Racial Groups; Sex Factors; Sleep; Sleep Wake Disorders

Flaxseeds have been known for their anti-cancerous effects due to the high abundance of lignans released upon ingestion. The most abundant lignan, secoisolariciresinol diglucoside (SDG), is ingested during the dietary intake of flax, and is then metabolized in the gut into two mammalian lignan derivatives, Enterodiol (END) and Enterolactone (ENL). These lignans were previously reported to possess anti-tumor effects against breast, colon, and lung cancer. This study aims to investigate the potential anti-cancerous effect of the flaxseed lignans SDG, END and ENL on acute myeloid leukemia cells (AML) in vitro and to decipher the underlying molecular mechanism. AML cell lines, (KG-1 and Monomac-1) and a normal lymphoblastic cell line were cultured and treated with the purified lignans. ENL was found to be the most promising lignan, as it exhibits a significant selective dose- and time-dependent cytotoxic effect in both AML cell lines, contrary to normal cells. The cytotoxic effects observed were attributed to apoptosis induction, as revealed by an increase in Annexin V staining of AML cells with increasing ENL concentrations. The increase in the percentage of cells in the pre-G phase, in addition to cell death ELISA analysis, validated cellular and DNA fragmentation respectively. Analysis of protein expression using western blots confirmed the activation of the intrinsic apoptotic pathway upon ENL treatment. This was also accompanied by an increase in ROS production intracellularly. In conclusion, this study demonstrates that ENL has promising anti-cancer effects in AML cell lines in vitro, by promoting DNA fragmentation and the intrinsic apoptotic pathway, highlighting the protective health benefits of flax seeds in leukemia.

Topics: 4-Butyrolactone; Antineoplastic Agents, Phytogenic; Apoptosis; Butylene Glycols; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Child; Female; Flax; Glucosides; Humans; Leukemia, Myeloid, Acute; Lignans; Plant Extracts; Reactive Oxygen Species; Seeds

Enterolignans, i.e. enterodiol and enterolactone, are polyphenols derived from the microbial metabolism of dietary lignans. They are considered phytoestrogens because of their estrogenic/antiestrogenic activity, which confers them benefits to human health when they reach sufficient levels in plasma. Hence, there is a great interest in studying the bacteria involved in enterolignan production. In the present study, three bifidobacterial strains (Bifidobacterium bifidum INIA P466, Bifidobacterium catenulatum INIA P732 and Bifidobacterium pseudolongum INIA P2) were found capable of producing low levels of enterodiol (2-11 μM) from lignan extracts; while another one (Bifidobacterium pseudocatenulatum INIA P946) was found to produce an important increment of the lignan secoisolariciresinol (SECO). Subsequently, the three enterodiol-producing bifidobacteria and another three Lactobacillus strains previously identified as enterolignans producers (Lactobacillus gasseri INIA P508, Lactobacillus salivarius INIA P448 and Lb. salivarius INIA P183), were tested on pure lignans yielding both enterodiol and enterolactone from secoisolariciresinol (SECO), while they did not metabolised the other lignan tested (i.e. matairesinol). B. catenulatum INIA P732 and Lb. gasseri INIA P508 were the strains that transformed the greatest percentage of SECO, yielding enterolactone concentrations above 2 mM. In addition, the formation of the intermediate compound dihydroxyenterodiol was observed as part of SECO transformation by all the strains. In this work, we have demonstrated for the first time how strains of Bifidobacterium and Lactobacillus are capable of carrying out the complete enterolignan metabolism, transforming a purified lignan (SECO) into enterodiol and enterolactone. The isolation and characterization of bacteria able to metabolize lignans and produce enterolignans, especially belonging to Bifidobacterium and Lactobacillus genera, is of biotechnological interest, because of their potential application in functional foods and as probiotics.

Topics: 4-Butyrolactone; Bifidobacterium; Diet; Humans; Lactobacillus; Lignans

Enterodiol (END) is transformed by human intestinal bacteria from lignans contained in various whole-grain cereals, nuts, legumes, flaxseed, and vegetables. It is known to have several physiological effects, but its effects on mitogen-activated protein kinase (MAPK) signaling and apoptosis in colorectal cancer (CRC) cells have not yet been elucidated. We therefore investigated the effects of END on apoptosis in CRC cells and whether these effects are mediated via MAPK signaling.. Cell proliferation was decreased by END treatment in a time-dependent manner. In particular, END treatment resulted in an apoptosis rate of up to 40% in CT26 cells but showed no cytotoxicity toward RAW264.7 macrophages. Treatment with END also suppressed the migration of CRC cells in a concentration-dependent manner. The phosphorylation of extracellular signal-regulated kinase (ERK), jun N-terminal kinase (JNK), and p38 was down-regulated with END treatment. Furthermore, END decreased the expression levels of anti-apoptotic proteins in CRC cells.. Enterodiol inhibited the growth of CRC cells by controlling the MAPK signaling pathway involved in proliferation and apoptosis. These results demonstrate that END has an apoptotic effect in CRC cells. © 2018 Society of Chemical Industry.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Extracellular Signal-Regulated MAP Kinases; Humans; Lignans; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction

Pinoresinol is a dimer of two β-β'-linked coniferyl alcohol molecules. It is both a plant defense molecule synthesized through the shikimic acid pathway and a representative of several β-β-linked dimers produced during the microbial degradation of lignin in dead plant material. Until now, little has been known about the bacterial catabolism of such dimers. Here we report the isolation of the efficient (+)-pinoresinol-mineralizing

Topics: 4-Butyrolactone; Benzaldehydes; Calcification, Physiologic; Furans; Gastrointestinal Microbiome; Humans; Lignans; Lignin; Metabolic Networks and Pathways; Minerals; Pseudomonas

Flaxseed is the most common and rich dietary source of lignans and is an acceptable supply of energy for livestock. Flaxseed lignans are precursors of enterolignans, mainly enterolactone and enterodiol, produced by the rumen and intestinal microbiota of mammals and have many important biological properties as phytoestrogens. Potential food-drug interactions involving flaxseed may be relevant for veterinary therapy, and for the quality and safety of milk and dairy products. Our aim was to investigate a potential food-drug interaction involving flaxseed, to explore whether the inclusion of flaxseed in sheep diet affects concentration of the antimicrobial danofloxacin in milk.. Increased concentrations of enterodiol and enterolactone were observed in sheep plasma and milk after 2 weeks of flaxseed supplementation (P < 0.05). However, enterolactone and enterodiol conjugates were not detected in milk. Milk danofloxacin pharmacokinetics showed that area under the curve (AUC). These findings highlight an effect of flaxseed-enriched diets on the concentration of antimicrobials in ruminant's milk, revealing the potential of these modified diets for the control of residues of antimicrobial drugs in milk.

Topics: 4-Butyrolactone; Animal Feed; Animals; Anti-Bacterial Agents; Diet; Female; Flax; Fluoroquinolones; Food-Drug Interactions; Lignans; Milk; Seeds; Sheep

The interaction of enterodiol and the well-described polyphenol epigallocatechin gallate (EGCG) with hepatic membranes has been matter of interest in the last few years. On one hand, EGCG is only able to bind to the phospholipid polar head groups, as it has been already described in synthetic lipid bilayers and erythrocyte membranes but cannot get inserted into the hydrophobic core or be transported into the lumen of membrane vesicles. On the other, enterodiol has no interaction with non-energized membranes either, but it is able to interact and even be transported upon addition of ATP. In fact, the ATPase activity undergoes a twofold increase in the presence of enterodiol but not in the presence of EGCG. This is the first report on the transport of enterodiol by liver membranes, and it may help explain the rather high blood concentrations of this estrogenic enterolignan compared to EGCG, which is extensively metabolized by the intestine and the liver. The present results suggest that a fraction of enterodiol may escape the liver inactivation by being pumped out from the hepatocytes to the bloodstream.

Topics: Adenosine Triphosphatases; Animals; Catechin; Cell Membrane; Hydrophobic and Hydrophilic Interactions; Lignans; Lipid Bilayers; Liver; Polyphenols; Rats

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.

Topics: 3T3-L1 Cells; 4-Butyrolactone; Adipogenesis; Adipose Tissue; Animals; Anti-Obesity Agents; Blood Glucose; Body Weight; Diet, High-Fat; Dietary Supplements; Fatty Liver; Gene Expression; Insulin Resistance; Lignans; Lipid Metabolism; Lipids; Male; Metabolic Syndrome; Mice; Mice, Inbred C57BL; Obesity; Picea; Plant Extracts

Ovarian cancer is one of the three leading gynecological malignancies, characterized by insidious growth, highly frequent metastasis, and quick development of drug resistance. As a result, this disease has low 5-year survival rates. Estrogen receptor inhibitors were commonly used for the treatment, but only 7% to 18% of patients respond to anti-estrogen therapies. Therefore, more effective therapies to inhibit estrogen-related tumors are urgently needed. Recently, phytoestrogens, such as lignans with estrogen-like biological activities, have attracted attention for their potential effects in the prevention or treatment of estrogen-related diseases. Enterodiol (END) and enterolactone (ENL) are mammalian lignans, which can reduce the risk of various cancers. However, the effects of END and ENL on ovarian cancer are not adequately documented.. We used in vitro assays on the ES-2 cell line to evaluate the inhibiting effects of END and ENL on ovarian cancer cell proliferation, invasion and migration ability and in vivo xenograft experiments on nude mice to validate the anticancer effects of END and ENL.. The in vitro assays demonstrated that high-dose END and ENL could obviously inhibit ovarian malignant properties, including cancerous proliferation, invasion, and metastasis. Compared to END, ENL behaved in a better time-dose dependent manner on the cancer cells. The in vivo experiments showed that END (1 mg/kg), ENL (1 mg/kg) and ENL (0.1 mg/kg) suppressed tumor markedly, and there were statistically significant differences between the experimental and control groups in tumor weight and volume. Compared to END, which have serious side effects to the animals at high concentration such as 1 mg/kg, ENL had higher anticancer activities and less side effects in the animals than END at the same concentrations, so it would be a better candidate for drug development.. END and ENL both have potent inhibitory effects on ovarian cancer but ENL possesses a more effective anti-cancer capability and less side effects than END. Findings in this work provide novel insights into ovarian cancer therapeutics with phytoestrogens and encourage their clinical applications.

Topics: 4-Butyrolactone; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cell Survival; Female; Humans; Lignans; Mice, Inbred BALB C; Mice, Nude; Ovarian Neoplasms; Phytoestrogens; Tumor Burden; Wound Healing

Flaxseed is the richest source of the plant lignan secoisolariciresinol diglucoside, which is converted to the mammalian lignans enterolactone (EL) and enterodiol by the gut microbiota of ruminants and humans. Enterolactone has been associated with improved animal and human health due to its antioxidant and anticarcinogenic properties. The objective of this study was to determine the pharmacokinetics of EL in newborn dairy calves fed milk replacer or EL-enriched milk. We hypothesized that newborn Holstein calves fed EL-enriched milk would have greater area under the curve and plasma concentration of EL compared with those fed milk replacer. On d 5 of life, calves were administered 2 L of milk replacer (n = 10; low-EL treatment: 123 nmol/L of EL) or 2 L of EL-enriched milk (n = 10; high-EL treatment: 481 nmol/L of EL) during the morning feeding (0700 h). Blood samples were taken from the jugular vein before (0 h) and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, and 48 h after oral administration of treatments. The area under the curve for the plasma concentration of EL was analyzed according to the trapezoidal rule between 0 and 12 h after treatment administration, and it was greater in high- (26 nmol/L × h) than low-EL calves (4.30 nmol/L × h). Similarly, the maximum concentration of EL in plasma was greater in high- (5.06 nmol/L) versus low-EL calves (1.95 nmol/L). Furthermore, the time after treatment intake to reach maximum plasma concentration of EL was faster in high- (4.31 h) compared with low-EL (4.44 h) treatment. Calves were able to absorb EL, indicating that EL-enriched milk can potentially be used as source of EL to pre-weaned ruminants.

Topics: 4-Butyrolactone; Animal Feed; Animals; Animals, Newborn; Cattle; Diet; Humans; Lignans; Milk

Flaxseed is a rich source of α-linolenic acid and phytoestrogens, mainly lignans, whose metabolites (enterodiol and enterolactone) can affect estrogen functions. The present study evaluated the influence of dietary flaxseed supplementation on reproductive performance and egg characteristics (fatty acids, cholesterol, lignans and isoflavones) of 40 Hy-Line hens (20/group) fed for 23 weeks a control diet or the same diet supplemented with 10% of extruded flaxseed. The flaxseed diet had approximately three times the content of lignans (2608.54 ng/g) as the control diet, mainly secoisolariciresinol diglucoside (1534.24 v. 494.72 ng/g). When compared with the control group, hens fed flaxseed showed a similar deposition rate (72.0% v. 73.9%) and egg yield. Furthermore, there was no effect of flaxseed on the main chemical composition of the egg and on its cholesterol content. Estradiol was higher in the plasma of the control group (1419.00 v. 1077.01 pg/ml) probably due to the effect of flaxseed on phytoestrogen metabolites. The plasma lignans were higher in hens fed flaxseed, whereas isoflavones were lower, mainly due to the lower equol value (50.52 v. 71.01 ng/ml). A similar trend was shown in eggs: the flaxseed group had higher level of enterodiol and enterolactone, whereas the equol was lower (198.31 v. 142.02 ng/g yolk). Secoisolariciresinol was the main lignan in eggs of the flaxseed group and its concentration was three times higher then control eggs. Flaxseed also improved the n-3 long-chain polyunsaturated fatty acids of eggs (3.25 v. 0.92 mg/g egg), mainly DHA, however, its oxidative status (thiobarbituric reactive substances) was negatively affected. In conclusion, 10% dietary flaxseed did not affect the productive performance of hens or the yolk cholesterol concentration, whereas the lignans and n-3 polyunsaturated fatty acid content of eggs improved. Further details on the competition between the different dietary phytoestrogens and their metabolites (estrogen, equol, enterodiol and enterolactone) should be investigated.

Topics: 4-Butyrolactone; alpha-Linolenic Acid; Animal Feed; Animals; Butylene Glycols; Chickens; Cholesterol; Diet; Dietary Supplements; Eggs; Fatty Acids; Fatty Acids, Omega-3; Female; Flax; Isoflavones; Lignans; Phytoestrogens; Seeds

Mammalian lignans or enterolignans are metabolites of plant lignans, an important category of phytochemicals. Although they are known to be associated with estrogenic activity, cell signaling pathways leading to specific cell functions, and especially the differences among lignans, have not been explored. We examined the estrogenic activity of enterolignans and their precursor plant lignans and cell signaling pathways for some cell functions, cell cycle and chemokine secretion. We used DNA microarray-based gene expression profiling in human breast cancer MCF-7 cells to examine the similarities, as well as the differences, among enterolignans, enterolactone and enterodiol, and their precursors, matairesinol, pinoresinol and sesamin. The profiles showed moderate to high levels of correlation (R values: 0.44 to 0.81) with that of estrogen (17β-estradiol or E2). Significant correlations were observed among lignans (R values: 0.77 to 0.97), and the correlations were higher for cell functions related to enzymes, signaling, proliferation and transport. All the enterolignans/precursors examined showed activation of the Erk1/2 and PI3K/Akt pathways, indicating the involvement of rapid signaling through the non-genomic estrogen signaling pathway. However, when their effects on specific cell functions, cell cycle progression and chemokine (MCP-1) secretion were examined, positive effects were observed only for enterolactone, suggesting that signals are given in certain directions at a position closer to cell functions. We hypothesized that, while estrogen signaling is initiated by the enterolignans/precursors examined, their signals are differentially and directionally modulated later in the pathways, resulting in the differences at the cell function level.

Topics: 4-Butyrolactone; Cell Cycle; Dioxoles; Estrogens; Furans; Gene Expression Profiling; Humans; Lignans; MCF-7 Cells; Oligonucleotide Array Sequence Analysis; Signal Transduction

Equol is a metabolite of daidzein that is produced by intestinal microbiota. The oestrogenic activity of equol is stronger than daidzein. Equol-producing bacteria are believed to play an important role in the gut. The rod-shaped and Gram-positive anaerobic equol-producing intestinal bacterium Slackia TM-30 was isolated from healthy human faeces and its effects on urinary phyto-oestrogen, plasma and faecal lipids were assessed in adult mice.. The urinary amounts of equol in urine were significantly higher in mice receiving the equol-producing bacterium TM-30 (BAC) group than in the control (CO) group (P < 0.05). However, no significant differences were observed between the urinary amounts of daidzein, dihydrodaidzein, enterodiol, and enterolactone between the BAC and CO groups. No significant differences in the plasma lipids were observed between the two groups. The lipid content (% dry weight) in the faeces sampled on the final day of the experiment tended to be higher in the BAC group than in the CO group (P = 0.07).. Administration of equol-producing bacterium TM-30 affected the urinary amounts of phyto-oestrogens and the faecal lipid contents of mice. The equol-producing bacterium TM-30 likely influences the metabolism of phyto-oestrogen via changes in the gastrointestinal environment. © 2015 Society of Chemical Industry.

Topics: 4-Butyrolactone; Actinobacteria; Animals; Equol; Feces; Female; Gastrointestinal Microbiome; Humans; Isoflavones; Lignans; Lipids; Mice; Mice, Inbred ICR; Phytoestrogens

The interaction of polyphenolic molecules with human serum albumin (HSA) could lead to changes in the reactivity of the HSA Cys34 thiol group (HSA-SH). The influences of enterolactone (EL) and enterodiol (ED) binding on HSA-SH reactivity in fatty acid (FA)-free HSA, and in HSA with bound stearic acid (S) in S/HSA molar ratios of 1:1 and 4:1, were investigated by the determination of the pseudo first order rate constants (k') for the thiol reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). The binding affinities and binding sites of EL and ED were also determined, using fluorescence measurements of the intrinsic fluorescence of Trp214 and diazepam (binding site marker). EL and ED binding to HSA increased the reactivity of HSA-SH in all assayed HSA-enterolignan complexes by 9.1-33.1%. The strongest effects were obtained for FA-free HSA-enterolignan complexes. S modulated/reduced the effect of EL on HSA-SH reactivity, while its influence on the effect of ED was negligible. The binding of enterolignans to HSA was investigated: the binding constants were the highest for FA-free HSA (EL: 11.64 × 10(4) M(-1) and ED: 5.59 × 10(4) M(-1) at 37 °C) and the lowest for S/HSA 4:1-enterolignan complexes (EL: 2.43 × 10(4) M(-1) and ED: 1.92 × 10(4) M(-1)). When the S/HSA ratio was increased, the binding affinities and number of binding sites for EL and ED were decreased. At the same time, a high correlation between binding constants and increased Cys34 reactivity was found (r = 0.974). Competitive experiments using diazepam indicated that the binding of ED and of EL was located in the hydrophobic pocket of site II in HSA. Overall, it is evident that stearic acid could modulate the enterolignan effects on HSA-SH reactivity as well as their binding to HSA. This finding could be important for pharmacokinetics and the expression of enterolignan antioxidant effects in vivo after an intake of lignan rich food.

Topics: 4-Butyrolactone; Antioxidants; Binding Sites; Cysteine; Humans; Lignans; Protein Binding; Serum Albumin; Spectrometry, Fluorescence; Sulfhydryl Compounds

This study aimed to improve the knowledge of secoisolariciresinol diglucoside (SDG) transformation by human gut microbiota.. SDG-supplemented microbiota cultures were inoculated with the feces of five subjects. The same volunteers received a flaxseed supplement for 7 days. SDG metabolites in cultures, feces, and urine were monitored by LC-ESI-QTOF and LC-DAD. In all cultures, SDG was deglycosylated to secoisolariciresinol (SECO) within 12 h. SECO underwent successive dehydroxylations and demethylations yielding enterodiol (4-18% conversion) and enterolactone (0.2-6%) after 24 h. Novel intermediates related to SECO, matairesinol (MATA), and anhydrosecoisolariciresinol (AHS) were identified in fecal cultures. These metabolites were also found after flaxseed consumption in feces and urine (in approximate amounts between 0.01-47.03 μg/g and 0.01-13.49 μg/mL, respectively) in their native form and/or modified by phase II human enzymes (glucuronide, sulfate and sulfoglucuronide conjugates).. Derivatives of MATA and AHS are described for the first time as intermediates of SDG biotransformation by intestinal bacteria, providing a more comprehensive knowledge of lignan intestinal metabolism. The transformations observed in vitro seem to occur in vivo as well. The detection in urine of SDG intermediates indicates their gut absorption, opening new perspectives on the study of their systemic biological effects.

Topics: 4-Butyrolactone; Adult; Bifidobacterium pseudocatenulatum; Butylene Glycols; Dietary Supplements; Feces; Female; Flax; Furans; Gastrointestinal Microbiome; Glucosides; Humans; Intestinal Mucosa; Intestines; Lignans; Male; Middle Aged; Prebiotics; Probiotics; Young Adult

The main scope of the present study was to analyze the membrane interaction of members of different classes of polyphenols, i.e. resveratrol, naringenin, epigallocatechin gallate and enterodiol, in model systems of different compositions and phase states. In addition, the possible association between membrane affinity and membrane protection against both lipid oxidation and bilayer-disruptive compounds was studied. Gibbs monolayer experiments indicated that even though polyphenols showed poor surface activity, it readily interacted with lipid films. Actually, a preferential interaction with expanded monolayers was observed, while condensed and cholesterol-containing monolayers decreased the affinity of these phenolic compounds. On the other hand, fluorescence anisotropy studies showed that polyphenols were able to modulate membrane order degree, but again this effect was dependent on the cholesterol concentration and membrane phase state. In fact, cholesterol induced a surface rather than deep into the hydrophobic core localization of phenolic compounds in the membranes. In general, the polyphenolic molecules tested had a better antioxidant activity when they were allowed to get inserted into the bilayers, i.e. in cholesterol-free membranes. On the other hand, a membrane-protective effect against bilayer permeabilizing activity of lysozyme, particularly in the presence of cholesterol, could be assessed. It can be hypothesized that phenolic compounds may protect membrane integrity by loosely covering the surface of lipid vesicles, once cholesterol push them off from the membrane hydrophobic core. However, this cholesterol-driven distribution may lead to a reduced antioxidant activity of linoleic acid double bonds.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Antioxidants; Catechin; Cholesterol; Dimyristoylphosphatidylcholine; Flavanones; Fluorescence Polarization; Hydrophobic and Hydrophilic Interactions; Lignans; Linoleic Acid; Lipid Bilayers; Lipid Peroxidation; Liposomes; Muramidase; Reactive Oxygen Species; Resveratrol; Stilbenes; Surface Properties

Hereinwe investigated the effect of elderflower extracts (EFE) and of enterolactone/enterodiol on hormone production and proliferation of trophoblast tumor cell lines JEG-3 and BeWo, as well as MCF7 breast cancer cells. The EFE was analyzed by mass spectrometry. Cells were incubated with various concentrations of EFE. Untreated cells served as controls. Supernatants were tested for estradiol production with an ELISA method. Furthermore, the effect of the EFE on ER/ER/PR expression was assessed by immunocytochemistry. EFE contains a substantial amount of lignans. Estradiol production was inhibited in all cells in a concentration-dependent manner. EFE upregulated ER in JEG-3 cell lines. In MCF7 cells, a significant ER downregulation and PR upregulation were observed. The control substances enterolactone and enterodiol in contrast inhibited the expression of both ER and of PR in MCF7 cells. In addition, the production of estradiol was upregulated in BeWo and MCF7 cells in a concentration dependent manner. The downregulating effect of EFE on ER expression and the upregulation of the PR expression in MFC-7 cells are promising results. Therefore, additional unknown substances might be responsible for ER downregulation and PR upregulation. These findings suggest potential use of EFE in breast cancer prevention and/or treatment and warrant further investigation.

Topics: 4-Butyrolactone; Cell Line, Tumor; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Estradiol; Female; Humans; Immunohistochemistry; Lignans; MCF-7 Cells; Phytoestrogens; Plant Extracts; Pregnancy; Progesterone; Receptors, Estrogen; Sambucus; Trophoblastic Neoplasms; Uterine Neoplasms

Lignans in plant foods are metabolized by gut bacteria to the enterolignans, enterodiol (END) and enterolactone (ENL). Enterolignans have biologic activities important to the prevention of cancer and chronic diseases. We examined the composition of the gut microbial community (GMC) as a contributor to human enterolignan exposure.. We evaluated the association between the GMC in stool, urinary enterolignan excretion, and diet from a 3-day food record in 115 premenopausal (ages 40-45 years) women in the United States. Urinary enterolignans were measured using gas chromatography-mass spectroscopy. The GMC was evaluated using 454 pyrosequencing of the 16S rRNA gene. Sequences were aligned in SILVA (www.arb-silva.de). Operational taxonomic units were identified at 97% sequence similarity. Taxonomic classification was performed and alpha and beta diversity in relationship to ENL production were assessed. Multivariate analysis and regression were used to model the association between enterolignan excretion and the GMC. Bacteria associated with ENL production were identified using univariate analysis and ridge regression.. After adjusting for dietary fiber intake and adiposity, we found a significant positive association between ENL excretion and either the GMC (P = 0.0007), or the diversity of the GMC (P = 0.01). The GMC associated with high ENL production was distinct (UNIFRAC, P < 0.003, MRPP) and enriched in Moryella spp., Acetanaerobacterium spp., Fastidiosipila spp., and Streptobacillus spp.. Diversity and composition of the GMC are associated with increased human exposure to enterolignans.. Differences in gut microbial diversity and composition explain variation in gut metabolic processes that affect environmental exposures and influence human health. Cancer Epidemiol Biomarkers Prev; 24(3); 546-54. ©2014 AACR.

Topics: 4-Butyrolactone; Adult; Diet Records; Female; Gastrointestinal Tract; Humans; Lignans; Microbiota; Middle Aged; Phenotype; Premenopause; United States

Consumption of flaxseed lignans is associated with various health benefits; however, little is known about the bioavailability of purified lignans in flaxseed. Data on their bioavailability and hence pharmacokinetics (PK) are necessary to better understand their role in putative health benefits. In the present study, we conducted a comparative PK analysis of the principal lignan of flaxseed, secoisolariciresinol diglucoside (SDG), and its primary metabolites, secoisolariciresinol (SECO), enterodiol (ED) and enterolactone (EL) in rats. Purified lignans were intravenously or orally administered to each male Wistar rat. SDG and its primary metabolites SECO, ED and EL were administered orally at doses of 40, 40, 10 and 10 mg/kg, respectively, and intravenously at doses of 20, 20, 5 and 1 mg/kg, respectively. Blood samples were collected at 0 (pre-dose), 5, 10, 15, 20, 30 and 45 min, and at 1, 2, 4, 6, 8, 12 and 24 h post-dosing, and serum samples were analysed. PK parameters and oral bioavailability of purified lignans were determined by non-compartmental methods. In general, administration of the flaxseed lignans SDG, SECO and ED demonstrated a high systemic clearance, a large volume of distribution and short half-lives, whereas administration of EL at the doses of 1 mg/kg (intravenously) and 10 mg/kg (orally administered) killed the rats within a few hours of dosing, precluding a PK analysis of this lignan. PK parameters of flaxseed lignans exhibited the following order: systemic clearance, SDG < SECO < ED; volume of distribution, SDG < SECO < ED; half-life, SDG < ED < SECO. The percentage of oral bioavailability was 0, 25 and < 1 % for SDG, SECO and ED, respectively.

Topics: 4-Butyrolactone; Administration, Oral; Animals; Biological Availability; Butylene Glycols; Dietary Supplements; Dose-Response Relationship, Drug; Estrogens; Flax; Glucosides; Half-Life; Injections, Intravenous; Intestinal Absorption; Kinetics; Lignans; Male; Metabolic Clearance Rate; Phytoestrogens; Random Allocation; Rats, Wistar; Seeds

The objective of the present study was to find the optimum dose of flaxseed that would decrease PG and alter oestrogen pathway endpoints implicated in ovarian cancer. In the study, four groups of fifty 1.5-year-old chickens were fed different amounts of flaxseed (0, 5, 10 or 15% of their total diet) for 4 months and were then killed to collect blood and tissues. Levels of flaxseed lignan metabolites, Enterolactone (EL) and Enterodiol (ED) were measured in the serum, liver and ovaries by liquid chromatography-MS/MS, and n-3 and n-6 fatty acid (FA) levels were measured by GC. The effects of the varied flaxseed doses were assessed by measuring levels of PGE2 and oestrogen metabolites (16-hydroxyestrone (16-OHE1) and 2-hydroxyestrone (2-OHE1)) as well as by analysing the expression of the oestradiol metabolising enzymes CYP3A4 (cytochrome p450, family 3, subfamily A, polypeptide 4), CYP1B1 (cytochrome p450, family 1, subfamily B, polypeptide 1) and CYP1A1 (cytochrome p450, family 1, subfamily A, polypeptide 1) and that of oestrogen receptor α (ERα) in the ovaries. The ratio of n-3:n-FA increased with an increase in flaxseed supplementation and corresponded to a dose-dependent decrease in cyclo-oxygenase-2 protein and PGE2 levels. EL and ED increased in the serum, liver and ovaries with increased concentrations of flaxseed. Flaxseed decreased the expression of ERα in the ovaries. The ratio of 2-OHE1:16-OHE1 in the serum increased significantly in the 15% flaxseed diet, and there was a corresponding increase in CYP1A1 in the liver and decrease in CYP3A4 in the ovaries. CYP1B1 mRNA also decreased with flaxseed diet in the ovaries. The 15% flaxseed-supplemented diet significantly decreased inflammatory PGE2, ERα, CYP3A4, CYP1B1 and 16-OHE1, but it increased CYP1A1 and 2-OHE1, which thus reduced the inflammatory and pro-carcinogenic micro-environment of the ovaries.

Topics: 4-Butyrolactone; Animals; Anticarcinogenic Agents; Chickens; Cyclooxygenase 1; Cyclooxygenase 2; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Cytochrome P-450 CYP3A; Diet; Dietary Supplements; Dinoprostone; Estrogen Receptor alpha; Estrogens; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Female; Flax; Hydroxyestrones; Lignans; Liver; Ovarian Neoplasms; Ovary; RNA, Messenger

Results from animal studies have consistently suggested that lignans play a role in the regulation of in body weight, but evidence from human studies has been limited. We examined the associations between urinary excretion of enterolactone and enterodiol, the major intestinal microbial metabolites of dietary lignans, and 10-year prospective weight change using data from 2 well-characterized cohort studies of US women: the Nurses' Health Study (2000-2010) and Nurses' Health Study II (1997-2007). Urinary excretion levels of enterolactone and enterodiol were measured at baseline. Associations with prospective weight change were analyzed using a multivariable-adjusted linear mixed-effects model. We observed that women in the highest quartile of urinary excretion of total lignans had significantly lower baseline body mass indices (weight in kilograms divided by height in meters squared) (mean, 24.6, 95% confidence interval (CI): 23.9, 25.2) than did those in the lowest quartile (mean, 27.7, 95% CI: 27.0, 28.4; P for trend < 0.01). Compared with women in the lowest quartile of enterodiol excretion, those in the highest quartile gained 0.27 kg/year less weight (95% CI: 0.12, 0.41; P for trend < 0.01) during the 10-year follow-up. The association was borderline significant for enterolactone (for the fourth vs. first quartile, least square mean of weight change rate = -0.14 kg/year, 95% CI: -0.29, 0.00). Our data suggest that higher urinary excretion of lignan metabolites, especially enterodiol, is associated with modestly slower weight gain.

Topics: 4-Butyrolactone; Adult; Aged; Body Weight; Diabetes Mellitus, Type 2; Female; Follow-Up Studies; Humans; Incidence; Lignans; Middle Aged; Prospective Studies; United States; Urinalysis

Flaxseed is an important source of lignan secoisolariciresinol diglucoside (SDG) and its aglycone, secoisolariciresinol (SECO). These phenolic compounds can be metabolized to the mammalian lignans enterodiol (ED) and enterolactone (EL) by human intestinal microflora. Flaxseed lignans are known for their potential health benefits, which are attributed to their antioxidant and phytoestrogenic properties. The focus of this study was to determine the bioaccessibility of plant and mammalian lignans in whole flaxseed (WF) and flaxseed flour (FF) throughout the entire digestive process. Moreover, the metabolic activity of intestinal microflora was evaluated.. A single-batch in vitro simulation of the digestive process was performed, including fermentation by the intestinal microflora in the colon. Bioaccessibility was calculated as (free lignan)/(total lignan). In digested WF, the bioaccessibility values of SECO, ED and EL were 0.75%, 1.56% and 1.23%, respectively. Conversely, in digested FF, the bioaccessibility values of SDG, ED and EL were 2.06%, 2.72% and 1.04%, respectively. The anaerobic count and short-chain fatty acids indicate that bacteria survival and carbohydrate fermentation occurred.. The contents of both SDG and ED were significantly higher in digested FF than in digested WF. FF facilitated the action of intestinal bacteria to release SDG and metabolize ED.

Topics: 4-Butyrolactone; Bacteria, Anaerobic; Biological Availability; Butylene Glycols; Colon; Fatty Acids, Volatile; Fermentation; Flax; Glucosides; Humans; In Vitro Techniques; Lignans; Seeds

Phytoestrogens have been associated with subtle hormonal changes, although effects on fecundity are unknown. Our objective was to evaluate the association between male and female urinary phytoestrogen (isoflavone and lignan) concentrations and time to pregnancy (TTP) in a population-based cohort of 501 couples desiring pregnancy and discontinuing contraception. Couples were followed for 12 mo or until pregnancy. Fecundability ORs (FORs) and 95% CIs were estimated after adjusting for age, body mass index, race, site, creatinine, supplement use, and physical activity in relation to female, male, and joint couple concentrations. Models included the phytoestrogen of interest and the sum of the remaining individual phytoestrogens. FORs <1 denote a longer TTP and FORs >1 a shorter TTP. Urinary lignan concentrations were higher, on average, among female partners of couples who became pregnant during the study compared with women who did not become pregnant (median enterodiol: 118 vs. 80 nmol/L; P < 0.10; median enterolactone: 990 vs. 412 nmol/L; P < 0.05) and were associated with significantly shorter TTP in models based on both individual and couples' concentrations (couples' models: enterodiol FOR, 1.13; 95% CI: 1.02, 1.26; enterolactone FOR, 1.11; 95% CI: 1.01, 1.21). Male lignan concentrations were not associated with TTP, nor were isoflavone concentrations. Sensitivity analyses showed that associations observed are unlikely to be explained by potential unmeasured confounding by lifestyle or other nutrients. Our results suggest that female urinary lignan concentrations at levels characteristic of the U.S. population are associated with a shorter TTP among couples who are attempting to conceive, highlighting the importance of dietary influences on fecundity.

Topics: 4-Butyrolactone; Adolescent; Adult; Body Mass Index; Diet; Female; Humans; Isoflavones; Lignans; Male; Phytoestrogens; Pregnancy; Prospective Studies; Socioeconomic Factors; Time-to-Pregnancy; Young Adult

Reports in the literature associate the dietary intake of flaxseed lignans with a number of health benefits. The major lignan found in flaxseed, secoisolariciresinol diglucoside (1), undergoes metabolism principally to secoisolariciresinol (2), enterodiol (3), and enterolactone (4) in the human gastrointestinal tract. Systemically, lignans are present largely as phase II enzyme conjugates. To improve understanding of the oral absorption characteristics, a systematic evaluation of the intestinal permeation was conducted and the conjugative metabolism potential of these lignans using the polarized Caco-2 cell system was analyzed. For permeation studies, lignans (100 μM) were added to acceptor or donor compartments and samples were taken at 2 h. For metabolism studies, lignans (100 μM) were incubated in Caco-2 for a maximum of 48 h. Cell lysates and media were treated with β-glucuronidase/sulfatase, and lignan concentrations were determined using HPLC. Apical-to-basal permeability coefficients for 2-4 were 8.0 ± 0.4, 7.7 ± 0.2, and 13.7 ± 0.2 (×10(-6)) cm/s, respectively, whereas efflux ratios were 0.8-1.2, consistent with passive diffusion. The permeation of compound 1 was not detected. The extent of conjugation after 48 h was <3%, ∼95%, ∼90%, and >99% for 1-4, respectively. These data suggest 2-4, but not 1 undergo passive permeation and conjugative metabolism by Caco-2 cells.

Topics: 4-Butyrolactone; Algorithms; Butylene Glycols; Caco-2 Cells; Chromatography, High Pressure Liquid; Flax; Glucosides; Humans; Intestinal Mucosa; Lignans; Molecular Structure; Permeability

Chronic inflammation has been implicated in the etiology of various chronic diseases. We previously found that certain urinary isoflavones are associated with markers of inflammation. In the present study, we examined the associations of serum C-reactive protein (CRP) and white blood cell (WBC) count with lignans, which are more frequent in the Western diet than isoflavones.. Our analysis included 2,028 participants of NHANES 2005-2008 and 2,628 participants of NHANES 1999-2004 aged 18 years and older. The exposures of interest were urinary mammalian lignans (enterodiol and enterolactone). Outcome variables were two inflammatory markers (CRP [≤10 mg/L] and WBC [≥3.0 and ≤11.7 (1,000 cells/μL)]). Log-transformed CRP concentration and WBC count by log-transformed creatinine-standardized concentrations of mammalian lignans were used for linear regression.. Statistically significant inverse associations of urinary lignan, enterodiol, and enterolactone concentrations with circulating CRP and WBC counts were observed in the multivariate-adjusted models: In NHANES 2005-2008, per one-percent increase in lignan concentrations in the urine, CRP concentrations and WBC counts decreased by 8.1 % (95 % CI -11.5, -4.5) and 1.9 % (95 % CI -2.7; -1.2), respectively. Per one-percent increase in enterodiol and enterolactone, WBC counts decreased by 2.1 % (95 % CI -2.8, -1.3) and 1.3 % (95 % CI -1.9, -0.6), respectively. In NHANES 1999-2004, analogous results were 3.0 % (95 % CI -5.6, -0.3), 1.2 % (95 % CI -2.0; -0.4), 1.0 % (95 % CI -1.8, -0.2), and 0.8 % (95 % CI -1.4, 0.2).. Mammalian lignans were inversely associated with markers of chronic inflammation. Due to the cross-sectional design, our findings require confirmation in prospective studies.

Topics: 4-Butyrolactone; Adult; Aged; Biomarkers; C-Reactive Protein; Chronic Disease; Cross-Sectional Studies; Female; Humans; Inflammation; Leukocyte Count; Lignans; Male; Middle Aged; Nutrition Surveys; United States

To examine urinary levels of enterolactone and enterodiol, intestinal microbial metabolites of dietary lignans, in relation to type 2 diabetes (T2D) risk.. Urinary concentrations of the lignan metabolites were assayed by liquid chromatography-mass spectrometry among 1,107 T2D and 1,107 control subjects in a nested case-control study conducted in participants from the Nurses' Health Study (NHS) and NHSII. Subjects were free of diabetes, cardiovascular disease, and cancer at urine sample collection in 1995-2001. Incident self-reported T2D cases identified through 2008 were confirmed with a validated questionnaire.. In both cohorts, T2D subjects had significantly lower concentrations of both enterolactone and enterodiol than control subjects. After multivariate adjustment for lifestyle and dietary risk factors of T2D, urinary concentrations of enterolactone were significantly associated with a lower risk of T2D (pooled odds ratio [OR] comparing the extreme quartiles 0.62 [95% CI 0.44, 0.88], P for trend = 0.003). Higher urinary concentrations of enterodiol were also marginally significantly associated with a lower T2D risk (pooled OR comparing extreme quartiles 0.67 [95% CI 0.48, 0.96], P for trend = 0.08). When concentrations of both metabolites were combined to reflect total lignan intake, the OR was 0.70 (95% CI 0.53, 0.92) for each SD increment of total lignan metabolites.. These results indicate that lignan metabolites, especially enterolactone, are associated with a lower risk of T2D in U.S. women. Further studies are needed to replicate these findings and to explore potential mechanisms underlying the observed association.

Topics: 4-Butyrolactone; Adult; Case-Control Studies; Diabetes Mellitus, Type 2; Diet; Female; Humans; Lignans; Microbiota; Middle Aged; Prospective Studies; Risk Factors

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).. 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.. 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.. The consumption of sesame and flaxseed may be beneficial to decrease metabolic parameters that are generally altered in MetS.

Topics: 3T3-L1 Cells; 4-Butyrolactone; Adipocytes; Adipose Tissue; Adiposity; Animals; Cell Differentiation; Diet, High-Fat; Dietary Fats; Disease Models, Animal; Insulin Resistance; Lignans; Linseed Oil; Male; Metabolic Syndrome; Mice; Mice, Inbred C57BL; Sesame Oil

The study objective was to evaluate independent and interactive associations of dietary fiber intake and high urinary enterolignans with cardiometabolic risk factors. The analysis included 2260 adults (≥20 y of age) from the 2003-2010 NHANES. Logistic regression models were used to evaluate obesity and clinically defined cardiometabolic risk factors in relation to dietary fiber intake and urinary enterolignan concentrations. Three sets of models were created: 1) independent associations, 2) mutually adjusted associations, and 3) interactions. Models were adjusted for age, gender, race/ethnicity, education, smoking status, and energy intake. High concentrations were considered to be above the 90th percentile of urinary enterolignan concentrations. Increasing dietary fiber intake was associated with high blood pressure (P = 0.02) and low serum HDL cholesterol (P-trend = 0.03). High urinary enterodiol concentration was not associated with obesity or cardiometabolic risk factors. High urinary enterolactone concentration was inversely associated with obesity (OR: 0.44; 95% CI: 0.29, 0.66), abdominal obesity (OR: 0.58; 95% CI: 0.39, 0.87), high serum C-reactive protein (CRP; OR: 0.52; 95% CI: 0.37, 0.74), high serum triglycerides (OR: 0.39; 95% CI: 0.23, 0.61), low serum HDL cholesterol (OR: 0.37; 95% CI: 0.23, 0.61), and metabolic syndrome (OR: 0.47; 95% CI: 0.30, 0.74). In mutually adjusted models, enterolactone associations observed in independent models remained similar, but associations for dietary fiber intake were attenuated, with the exception of blood pressure. In interaction models, there were 2 significant interactions: between high urinary enterodiol concentration and dietary fiber intake for high serum CRP (P = 0.04) and high plasma glucose (P = 0.04). Overall, being in the highest 10% of urinary enterolactone concentration was associated with cardiometabolic risk factors, independent of dietary fiber intake and enterodiol concentration. Future studies are warranted to evaluate physiologic actions of enterolactone or aspects of the gut microbial profile responsible for lignan metabolism to enterolactone.

Topics: 4-Butyrolactone; Adult; Aged; Aged, 80 and over; Blood Glucose; Blood Pressure; C-Reactive Protein; Cardiovascular Diseases; Cholesterol, HDL; Dietary Fiber; Female; Humans; Lignans; Logistic Models; Male; Metabolic Syndrome; Middle Aged; Nutrition Surveys; Obesity; Odds Ratio; Phytoestrogens; Risk Factors; Triglycerides

Sesaminol triglucoside is a major lignin in sesame meal and has a methylenedioxyphenyl group and multiple functions in vivo. As a tetrahydrofurofuran type lignan, sesaminol triglucoside is metabolized to mammalian lignans. This investigation studies the effect of sesaminol triglucoside and its tetrahydrofuranoid metabolites (sesaminol, 2-episesaminol, hydroxymethyl sesaminol-tetrahydrofuran, enterolactone, and enterodiol) on gene expression in primary rat hepatocytes using a DNA microarray. Sesame lignans significantly affected the expression of xenobiotic-induced transcripts of cytochrome P450, solute carrier (SLC), and ATP-binding cassette (ABC) transporters. Changes in gene expression were generally greater in response to metabolites with methylenedioxyphenyl moieties (sesaminol triglucoside, sesaminol, and 2-episesaminol) than to the tetrahydrofuranoid metabolites (hydroxymethyl sesaminol-tetrahydrofuran, enterolactone, and enterodiol). Tetrahydrofuran lignans, such as sesaminol triglucoside, sesamin, hydroxymethyl sesaminol-tetrahydrofuran, and sesaminol changed the expression of ABC transporters.

Topics: 4-Butyrolactone; Animals; ATP-Binding Cassette Transporters; Cytochrome P-450 Enzyme System; Dioxoles; Furans; Gene Expression; Gene Expression Profiling; Glucosides; Hepatocytes; Inactivation, Metabolic; Lignans; Liver; Mammals; Plant Extracts; Rats, Sprague-Dawley; Seeds; Sesamum

The gut microbial environment (bacteria and metabolites) may have a role in obesity. Urinary enterolignan concentrations can provide a marker of interindividual differences in microbial environments. Analysis was conducted on 6806 individuals from 2003 to 2008 National Health and Nutrition Examination Survey data, and weight status, waist circumference groups and high- vs low-enterolignan concentration was evaluated using multinomial logistic regression, adjusted for personal and dietary factors. High-enterolignan concentrations were defined as the 90th percentile value and greater. High-enterodiol concentration was associated with 18% and 42% lower likelihood of being overweight and obese, respectively, and 48% lower likelihood of having high-risk waist circumference among adults. High-enterolactone concentration was associated with 24% and 64% lower likelihood of being overweight and obese, respectively. Age and sex were not modifiers of these associations. These results from a large human study population provide additional evidence supporting the microbiome-obesity relationship.

Topics: 4-Butyrolactone; Adolescent; Adult; Bacteria; Biomarkers; Child; Female; Gastrointestinal Tract; Humans; Lignans; Logistic Models; Male; Microbiota; Obesity; Overweight; Risk Factors; Waist Circumference

Phytoestrogens are plant-derived, non-steroidal phytochemicals with anticarcinogenic potential. The major structural classes are the isoflavones and lignans. The aim of this study was to compare the effect of the plant-derived lignans secoisolariciresinol and matairesinol with the human lignans enterodiol and enterolactone as well as with 17β estradiol and tamoxifen on cell proliferation of breast carcinoma cell lines.. The influence of the lignans, 17β estradiol and tamoxifen on cell proliferation was determined using the BrdU test in MCF 7 and BT 20 cell lines.. Enterodiol and enterolactone induced a stronger inhibition of cell growth in MCF 7 and BT 20 cells than secoisolariciresinol and matairesinol. The inhibition effects were less expressed in the BT 20 than in the MCF 7 cells.. The human lignans enterodiol and enterolactone are more biologically active than their precursors secoisolariciresinol and matairesinol, and may be defined as the real drugs in cancer prevention.

Topics: 4-Butyrolactone; Anticarcinogenic Agents; Breast Neoplasms; Butylene Glycols; Cell Line, Tumor; Cell Proliferation; Estradiol; Estrogens; Female; Furans; Humans; Lignans; Phytoestrogens; Selective Estrogen Receptor Modulators; Tamoxifen

Fiber-rich diets are associated with favorable lipid profiles, but the specific compounds and the mechanisms behind this effect are yet to be fully understood. Lignans are fiber-related polyphenols that have been associated with lower prevalence of cardiovascular disease. The objective of this study was to investigate the relationship between dietary lignan exposure, measured as the urinary concentration of their metabolites, enterolactone and enterodiol, and serum lipids in a representative sample of U.S. adults. We carried out a cross-sectional analysis of data from 1492 adults who participated in the 1999-2004 NHANES. The mean urinary concentration of enterolignans in U.S. adults was 1.9 μmol/L. The multivariate-adjusted mean differences comparing the highest and lowest enterolignan tertile were 0.06 mmol/L for HDL cholesterol and -0.17 mmol/L for TG (P < 0.05). In spline regression models, we also found an inverse association between serum TG and urinary enterolignan concentrations. Serum HDL cholesterol levels increased linearly with increasing enterolignan concentrations. Modest or no associations were found between enterolignan concentrations and serum LDL cholesterol or total cholesterol. In conclusion, enterolignan concentrations are associated with lower serum TG concentrations and greater HDL cholesterol concentrations in U.S. adults.

Topics: 4-Butyrolactone; Adult; Aged; Aged, 80 and over; Biomarkers; Cardiovascular Diseases; Cholesterol, HDL; Cross-Sectional Studies; Dietary Fiber; Female; Humans; Lignans; Male; Middle Aged; Nutrition Surveys; Regression Analysis; Risk Factors; Triglycerides; United States; Young Adult

Phytoestrogens are structurally similar to estrogens and may affect breast cancer risk by mimicking estrogenic/antiestrogenic properties. In Western societies, whole grains and possibly soy foods are rich sources of phytoestrogens. A population-based case-control study in German postmenopausal women was used to evaluate the association of phytoestrogen-rich foods and dietary lignans with breast cancer risk. Dietary data were collected from 2,884 cases and 5,509 controls using a validated food-frequency questionnaire, which included additional questions phytoestrogen-rich foods. Associations were assessed using conditional logistic regression. All analyses were adjusted for relevant risk and confounding factors. Polytomous logistic regression analysis was performed to evaluate the associations by estrogen receptor (ER) status. High and low consumption of soybeans as well as of sunflower and pumpkin seeds were associated with significantly reduced breast cancer risk compared to no consumption (OR = 0.83, 95% CI = 0.70-0.97; and OR = 0.66, 95% CI = 0.77-0.97, respectively). The observed associations were not differential by ER status. No statistically significant associations were found for dietary intake of plant lignans, fiber, or the calculated enterolignans. Our results provide evidence for a reduced postmenopausal breast cancer risk associated with increased consumption of sunflower and pumpkin seeds and soybeans.

Topics: 4-Butyrolactone; Aged; Breast Neoplasms; Carcinoma; Case-Control Studies; Cucurbita; Diet; Dietary Fiber; Female; Germany; Glycine max; Helianthus; Humans; Lignans; Middle Aged; Phytoestrogens; Postmenopause; Risk; Seeds

Starting from previous structure-activity relationship studies of taste modifiers based on homoeriodictyol, dihydrochalcones, deoxybenzoins, and trans-3-hydroxyflavones as obvious analogues were investigated for their masking effect against caffeine. The most active compounds of the newly investigated taste modifiers were phloretin, the related dihydrochalcones 3-methoxy-2',4,4'-trihydroxydihydrochalcone and 2',4-dihydroxy-3-methoxydihydrochalcone, and the deoxybenzoin 2-(4-hydroxy-3-methoxyphenyl)-1-(4-hydroxyphenyl)ethanone. Starting with the whole set of compounds showing activity >22%, a (Q)SAR pharmacophore model for maskers of caffeine bitterness was calculated to explain the structural requirements. After docking of the pharmacophore into a structural model of the broadly tuned bitter receptor hTAS2R10 and docking of enterolactone and enterodiol as only very weakly related structures, it was possible to predict qualitatively their modulating activity. Enterodiol (25 mg L(-1)) reduced the bitterness of the 500 mg L(-1) caffeine solution by about 30%, whereas enterolactone showed no masking but a slight bitter-enhancing effect.

Topics: Adult; Caffeine; Female; Flavones; Humans; Lignans; Male; Middle Aged; Models, Molecular; Molecular Structure; Receptors, G-Protein-Coupled; Taste

Dietary lignans show some promising health benefits, but little is known about their fate and activities in the small intestine. The purpose of this study was thus to investigate whether plant lignans are taken up by intestinal cells and modulate the intestinal inflammatory response using the Caco-2 cell model. Six lignan standards [secoisolariciresinol diglucoside (SDG), secoisolariciresinol (SECO), pinoresinol (PINO), lariciresinol, matairesinol (MAT), and hydroxymatairesinol] and their colonic metabolites [enterolactone (ENL) and enterodiol] were studied. First, differentiated cells were exposed to SDG, SECO, PINO, or ENL at increasing concentrations for 4 h, and their cellular contents (before and after deconjugation) were determined by HPLC. Second, in IL-1β-stimulated confluent and/or differentiated cells, lignan effects were tested on different soluble proinflammatory mediators quantified by enzyme immunoassays and on the NF-κB activation pathway by using cells transiently transfected. SECO, PINO, and ENL, but not SDG, were taken up and partly conjugated by cells, which is a saturable conjugation process. PINO was the most efficiently conjugated (75% of total in cells). In inflamed cells, PINO significantly reduced IL-6 by 65% and 30% in confluent and differentiated cells, respectively, and cyclooxygenase (COX)-2-derived prostaglandin E(2) by 62% in confluent cells. In contrast, MAT increased significantly COX-2-derived prostaglandin E(2) in confluent cells. Moreover, PINO dose-dependently decreased IL-6 and macrophage chemoattractant protein-1 secretions and NF-κB activity. Our findings suggest that plant lignans can be absorbed and metabolized in the small intestine and, among the plant lignans tested, PINO exhibited the strongest antiinflammatory properties by acting on the NF-κB signaling pathway, possibly in relation to its furofuran structure and/or its intestinal metabolism.

Topics: 4-Butyrolactone; Anti-Inflammatory Agents; Butylene Glycols; Caco-2 Cells; Cell Differentiation; Chemokine CCL2; Chromatography, High Pressure Liquid; Cyclooxygenase 2; Furans; Glucosides; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Intestines; Lignans; NF-kappa B; Plant Extracts; Signal Transduction

To investigate the in vitro effect of the combination of lignan enterolactone (ENL) or lignan enterodiol (END) with melatonin on steroid hormone secretion and cellular aromatase content in human adrenal carcinoma cells.. Human adrenocortical carcinoma cells.. Melatonin plus ENL or END was added to cell culture medium along with cAMP (100μM); control cells received cAMP alone. Medium and cell lysates were collected after 24 and 48 hours of cultivation. Samples of medium were analyzed for progesterone, 17-hydroxyprogesterone, androstenedione, aldosterone, estradiol, and cortisol concentration by use of radioimmunoassays. Cell lysates were used for western blot analysis of aromatase content.. The addition of ENL or END with melatonin to cAMP-stimulated cells (treated cells) resulted in significant decreases in estradiol, androstenedione, and cortisol concentrations at 24 and 48 hours, compared with concentrations in cells stimulated with cAMP alone (cAMP control cells). The addition of these compounds to cAMP-stimulated cells also resulted in higher progesterone and 17-hydroxyprogesterone concentrations than in cAMP control cells; aldosterone concentration was not affected by treatments. Compared with the content in cAMP control cells, aromatase content in treated cells was significantly lower.. The combination of lignan and melatonin affected steroid hormone secretion by acting directly on adrenal tumor cells. Results supported the concept that this combination may yield similar effects on steroid hormone secretion by the adrenal glands in dogs with typical and atypical hyperadrenocorticism.

Topics: 4-Butyrolactone; Adrenal Glands; Animals; Aromatase; Cell Line; Cyclic AMP; Dogs; Humans; Lignans; Melatonin; Phytoestrogens; Steroids

Lignans are ubiquitous plant polyphenols, which have relevant health properties being the major phytoestrogens occurring in Western diets. Secoisolariciresinol (SECO) is the major dietary lignan mostly found in plants as secoisolariciresinol diglucoside (SDG). To exert biological activity, SDG requires being deglycosylated to SECO and transformed to enterodiol (ED) and enterolactone (EL) by the intestinal microbes. The involvement of bifidobacteria in the transformation of lignans glucosides has been investigated for the first time in this study. Twenty-eight strains were assayed for SDG and SECO activation. They all failed to transform SECO into reduced metabolites, excluding any role in ED and EL production. Ten Bifidobacterium cultures partially hydrolyzed SDG, giving both SECO and the monoglucoside with yields < 25%. When the cell-free extracts were assayed in SDG transformation, seven additional strains were active in the hydrolysis. Cellobiose induced β-glucosidase activity and caused the enhancement of both the rate of SDG hydrolysis and the final yield of SECO only in the strains capable of SDG bioconversion. The highest SDG conversion to SECO was achieved by Bifidobacterium pseudocatenulatum WC 401, which exhibited 75% yield in cellobiose-based medium after 48 h. These results indicate that SDG hydrolysis is not a common feature in Bifidobacterium genus, but selected probiotic strains can be combined to β-glucoside-based prebiotics to enhance the release of SECO, thus improving its bioavailability for absorption by colonic mucosa and/or the biotransformation to ED and EL by other intestinal microorganisms.

Topics: 4-Butyrolactone; Bifidobacterium; Biotransformation; Butylene Glycols; Glucosides; Lignans

The bioactivity of lignans depends on their transformation by gut bacteria. The intestinal bacteria Clostridium saccharogumia, Eggerthella lenta, Blautia producta and Lactonifactor longoviformis convert the plant lignan secoisolariciresinol diglucoside via secoisolariciresinol (SECO) into the bioactive enterolignans enterodiol (ED) and enterolactone (EL). While the in vitro conversion of lignans by these bacteria has already been demonstrated, it is unclear whether this defined community is also capable of catalysing lignan transformation in vivo. We therefore associated germ-free rats with these four species. Germ-free rats served as a control. All animals were fed a diet containing 5% ground flaxseed. The caecal contents of rats associated with the four lignan-activating bacteria (ALB rats) contained SECO, ED and EL. The maximal EL formation rate from lignans in the pooled caecal contents of ALB rats was 7.52 nmol min(-1) g(-1) dry matter. The ALB rats excreted EL, but no SECO and ED, in their urine. The caecal contents of germ-free rats contained SECO, but no ED and EL. Their urine was devoid of lignans. Hence, the presence of enterolignans in the ALB rats, but not in the germ-free rats, demonstrates that this defined microbial community is capable of transforming plant lignans into EL in vivo.

Topics: 4-Butyrolactone; Animals; Biotransformation; Butylene Glycols; Cecum; Diet; Feces; Flax; Germ-Free Life; Gram-Positive Bacteria; Lignans; Rats; Rats, Sprague-Dawley

The effects of enterolignans, e.g., enterodiol (END) and particularly its oxidation product, enterolactone (ENL), on prevention of hormone-dependent diseases, such as osteoporosis, cardiovascular diseases, hyperlipemia, breast cancer, colon cancer, prostate cancer and menopausal syndrome, have attracted much attention. To date, the main way to obtain END and ENL is chemical synthesis, which is expensive and inevitably leads to environmental pollution. To explore a more economic and eco-friendly production method, we explored biotransformation of enterolignans from precursors contained in defatted flaxseeds by human intestinal bacteria.. We cultured fecal specimens from healthy young adults in media containing defatted flaxseeds and detected END from the culture supernatant. Following selection through successive subcultures of the fecal microbiota with defatted flaxseeds as the only carbon source, we obtained a bacterial consortium, designated as END-49, which contained the smallest number of bacterial types still capable of metabolizing defatted flaxseeds to produce END. Based on analysis with pulsed field gel electrophoresis, END-49 was found to consist of five genomically distinct bacterial lineages, designated Group I-V, with Group I strains dominating the culture. None of the individual Group I-V strains produced END, demonstrating that the biotransformation of substrates in defatted flaxseeds into END is a joint work by different members of the END-49 bacterial consortium. Interestingly, Group I strains produced secoisolariciresinol, an important intermediate of END production; 16S rRNA analysis of one Group I strain established its close relatedness with Klebsiella. Genomic analysis is under way to identify all members in END-49 involved in the biotransformation and the actual pathway leading to END-production.. Biotransformation is a very economic, efficient and environmentally friendly way of mass-producing enterodiol from defatted flaxseeds.

Topics: Biotransformation; Butylene Glycols; Cluster Analysis; DNA Fingerprinting; DNA, Bacterial; DNA, Ribosomal; Electrophoresis, Gel, Pulsed-Field; Enterobacteriaceae; Feces; Female; Flax; Gastrointestinal Tract; Humans; Lignans; Male; Molecular Sequence Data; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Young Adult

Lignan-rich whole-grain cereals, beans, berries, and nuts show protective effects against a variety of chronic diseases, including cancer. Lignans are converted by intestinal microflora to enterolactone (EL) and its oxidation product enterodiol (ED). To investigate the immunomodulatory effect of EL and ED in human cells, peripheral blood lymphocytes were treated with increasing physiologically relevant concentrations of EL and ED (0-1000 microM) and stimulated with lipopolysaccharide (LPS) and anti-CD3 plus anti-CD28 monoclonal antibodies. A dose-related inhibition of cell proliferation and cytokine production was observed, with EL being the most active. Molecular investigations in THP-1 cells showed that both EL and ED prevented inhibitory-kappaB (I-kappaB) degradation and nuclear factor-kappaB (NF-kappaB) activation, which in turn resulted in decreased tumor necrosis factor-alpha (TNF-alpha) production. EL and ED were also able to pass the intestinal barrier and modulate cytokine production. The findings of the present study reveal potential mechanisms that could explain some in vivo beneficial effects of lignans.

Topics: 4-Butyrolactone; Cell Line; Cell Proliferation; Cells, Cultured; Humans; Immunologic Factors; Lignans; Lymphocytes; NF-kappa B; Signal Transduction; Tumor Necrosis Factor-alpha

A human intestinal bacterium, strain END-2, which enantioselectively oxidizes (+)-enterodiol (END) to (+)-enterolactone (ENL) through enterolactol is also responsible for demethylation during plant lignan metabolism. A free hydroxyl group adjacent to the methoxy group is required for demethylation. The bacterium transformed (+)- and (-)-secoisolariciresinol to (+)-ENL and (-)-END, respectively, by co-incubation with strain ARC-1, which is responsible for dehydroxylation.

Topics: 4-Butyrolactone; Bacteria, Anaerobic; Biotransformation; Butylene Glycols; Chromatography, High Pressure Liquid; Feces; Humans; Intestines; Lignans; Mass Spectrometry; Methylation; Molecular Structure; Stereoisomerism; Substrate Specificity

Plant lignans present in foods such as whole grains, seeds and nuts, fruits and vegetables, and beverages. Plant lignans are converted by intestinal bacteria into the enterolignans enterodiol and enterolactone. Up to now, epidemiological evidence for a protective role of enterolignans on cardiovascular diseases is limited and inconsistent. We investigated the association between plasma enterodiol and enterolactone and nonfatal myocardial infarction risk in a prospective study. During follow-up (1987-1998) of 15,107 subjects, aged 20-59 years, 236 incident nonfatal myocardial infarction cases were diagnosed. Controls (n=283) were frequency matched to the cases on age, sex, and study center. No statistically significant associations between plasma enterodiol and enterolactone and risk of nonfatal myocardial infarction were detected. The odds ratio for the highest versus the lowest quartile of enterodiol was 1.21 (95% confidence interval (CI): 0.70, 2.12; p for trend=0.74), and that of enterolactone 1.51 (95% CI: 0.87, 2.61; p for trend=0.12) after adjustment for known dietary risk factors for coronary heart disease. No effect modification was observed for sex, menopausal status, or smoking status. Our results do not support the hypothesis that high plasma enterodiol or enterolactone concentrations are associated with a reduced risk of nonfatal myocardial infarction.

Topics: 4-Butyrolactone; Adult; Antioxidants; Cardiovascular Diseases; Case-Control Studies; Female; Humans; Lignans; Male; Middle Aged; Myocardial Infarction; Prospective Studies; Risk; Treatment Outcome

A total of 24 lactating Holstein cows averaging 620 (SE=29) kg of body weight were allotted at week 17 of lactation to eight groups of three cows blocked for similar days in milk to determine the effects of feeding two sources of the plant lignan precursor secoisolariciresinol diglucoside, whole flaxseed and flaxseed meal, on concentrations of the mammalian lignans (enterodiol and enterolactone) in milk. Feed intake, digestion, milk production and milk composition were also determined to compare the use of whole flaxseed and flaxseed meal for milk production. Cows within each block were assigned to one of the three isonitrogenous and isoenergetic total mixed diets: no flaxseed product; 10% flaxseed meal; or 10% whole flaxseed in the dry matter. The experiment was carried out from week 17 to week 21 of lactation and diets were fed at ad-libitum intake. The mammalian lignan, enterodiol, was not detected in the milk of cows. Cows fed whole flaxseed and flaxseed meal had greater concentrations of enterolactone in milk than those fed the control diet. Feed intake, milk production and milk composition were also similar for all diets, indicating that both flaxseed meal and whole flaxseed are suitable feed ingredients for milk production of cows in mid lactation. The results provide new information on the conversion of plant secoisolariciresinol diglucoside from two flaxseed products into mammalian lignans in dairy cows.

Topics: 4-Butyrolactone; Animals; Butylene Glycols; Cattle; Diet; Digestion; Fatty Acids; Flax; Glucosides; Lactation; Lignans; Male; Milk; Seeds

Phytoestrogens (isoflavones, enterolignans and coumestrol) in wastewater samples and surface water samples have been analysed by LC-ESI-MS(n). In wastewater samples, high levels of enterolactone (581-2111 ng/L), daidzein (341-1688 ng/L) and enterodiol (60-834 ng/L) were detected in raw sewage, but the vast majority of the analysed phytoestrogens were removed effectively in the treatment process. The removal rates of the analysed phytoestrogens in the two advanced tertiary treatment plants were >99%; a case study in one of the treatment plants showed that most of the residual phytoestrogens were removed by biological treatment using activated sludge. In surface water samples, daidzein was found at concentrations ranging from 2 ng/L to 33 ng/L in samples from two creeks, and up to 120 ng/L in surface water (pond) on a dairy farm. The analytical results suggest that direct excretions of livestock discharged from farmyards can be another potential source of phytoestrogen contamination in the aquatic environment.

Topics: 4-Butyrolactone; Chromatography, Liquid; Coumestrol; Dairying; Environmental Monitoring; Fresh Water; Isoflavones; Lignans; Phytoestrogens; Spectrometry, Mass, Electrospray Ionization; Waste Disposal, Fluid; Water Pollutants, Chemical

The microbial metabolism of dietary phytoestrogens varies considerably among individuals and influences the final exposure to bioactive compounds. In view of the increasing number of food supplements combining several classes of phytoestrogens, the microbial potential to activate various proestrogens within an individual was evaluated in 3 randomized dietary crossovers. Treatment allocation was based on participants' eligibility (>45% in vitro bioactivation of >or=2 separate proestrogens by fecal cultures; n = 40/100). After a run-in of >or=4 d, participants were given soy-, hop-, and/or flax-based food supplements dosed either separately (SOY: 2.83 mg daidzein aglycone equivalents/supplement, HOP: 1.20 mg isoxanthohumol (IX)/supplement, or FLAX: 2.08 mg secoisolariciresinol (SECO) aglycone equivalents/supplement; reference intervention) or simultaneously (MIX; test intervention) 3 times/d for 5 d, followed by a wash-out period (>or=7 d) and the second intervention. Before and after each (co)supplementation, spot urine and serum were collected. In total, 22 equol, 19 8-prenylnaringenin (8-PN), and 21 enterolactone (ENL) producers completed the SOY+MIX, HOP+MIX, and FLAX+MIX trials, respectively. The microbial bioactivation of daidzein, IX, and SECO, generally decreased upon coincubation in vitro (equol: 4.4%, P = 0.164; 8-PN: 20.5%, P < 0.001; ENL: 44.3%, P < 0.001) and cosupplementation in vivo (equol: 28.3%, P = 0.009; 8-PN: 35.4%, P = 0.107; ENL: 35.9%, P = 0.003). Although the bioavailabilities of total isoflavones, prenylflavonoids, and lignans were not significantly affected upon coadministration, participants were exposed to lower phytoestrogen-derived 17beta-estradiol equivalents. In conclusion, the bioavailability of phytoestrogens, especially when given in mixtures, is subject to high interindividual variation. These findings support the importance of personalized screening when assessing the efficacy of such products and mixtures.

Topics: Dietary Supplements; Equol; Estradiol; Feces; Flavanones; Flavonoids; Genistein; Humans; Isoflavones; Lignans; Phytoestrogens

Plant lignans are converted by the intestinal microflora to the mammalian lignans enterodiol and enterolactone, which are associated with beneficial health effects in humans. The mammalian lignans enterodiol and enterolactone are believed to protect against certain diseases, e.g., breast and prostate cancer as well as coronary heart disease. In this study, the effects of enterodiol and enterolactone on osteoblastic differentiation were examined. It was found that enterolactone and enterodiol showed the same effects. They have biphasic effects on cell viability, alkaline phosphotase (ALP) activity, transcriptional level of osteonectin, and collagen I, showing induction at low doses and inhibition at high doses. They increased transcriptional levels of ALP, osteopontin, and osteocalcin in a dose-dependent manner. The difference may be related to the estrogenic and antiestrogenic character and multiple signaling transduction of phytoestrogen.

Topics: 4-Butyrolactone; Alkaline Phosphatase; Cell Differentiation; Cell Line; Cell Survival; Collagen Type I; Dose-Response Relationship, Drug; Humans; Lignans; Osteoblasts; Osteonectin; Reverse Transcriptase Polymerase Chain Reaction; RNA; Transcription, Genetic

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.

Topics: 4-Butyrolactone; 9,10-Dimethyl-1,2-benzanthracene; Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Apoptosis; Butylene Glycols; Carcinogens; Cell Proliferation; Dietary Supplements; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Furans; Gene Expression Regulation, Neoplastic; Humans; Lignans; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Ovariectomy; Phytoestrogens; Rats; Receptors, Progesterone; Transplantation, Heterologous

We hypothesized that the inclusion of flaxseed in the diets of lactating dairy cows will increase urinary and fecal concentrations of the lignans, secoisolariciresinol diglycoside (SDG), enterolactone and enterodiol, reduce intrafollicular concentrations of IGF-I and estradiol, and subsequently reduce estradiol and oxytocin receptor expression in the endometrium. To test this hypothesis, 27 cycling, lactating Holstein cows were assigned to 1 of 3 diets supplemented with saturated fatty acids (SAT), flax (FLX), or sunflower (SUN) seed. Rations were formulated to provide 750 g supplemental fat/cow/d in all dietary groups. Ovulation (Day 0) was synchronized, and 5 d later, follicles>8 mm were ablated by an ultrasound-guided procedure in all cows. Samples of blood (Days 0 to 14), follicular fluid (Day 5 and 15), endometrium (Day 15), as well as urine and feces were collected in a subset of the animals. The fecal concentrations of SDG and enterodiol were higher (P<0.05) in cows fed FLX than in those fed SAT or SUN. Enterodiol increased (P<0.05) in urine samples of cows fed FLX, compared to those of cows fed SUN. However, follicular estradiol concentrations on Day 5 and 15 and endometrial concentrations of estradiol and oxytocin receptors on Day 15 did not differ among the dietary groups. Mean plasma progesterone concentrations were higher (P<0.05) in cows fed FLX and SUN than in those fed SAT. In summary, a diet supplemented with flaxseed increased the concentrations of SDG and enterodiol in feces, as hypothesized, but did not alter intrafollicular concentrations of IGF-I or estradiol, or endometrial populations of oxytocin or estrogen receptors in lactating dairy cows.

Topics: Animals; Cattle; Diet; Dietary Fats; Endometrium; Estradiol; Feces; Female; Flax; Follicular Fluid; Helianthus; Hydrogenation; Lignans; Progesterone; Receptors, Estrogen; Receptors, Oxytocin; Seeds

To determine the in vitro conversion of plant lignans from two flax products (hull and seed) into the mammalian lignans, enterolactone and enterodiol, by bovine ruminal and faecal microbiota.. Flax seeds and hulls were incubated in vitro over a 96-h time course with ruminal or faecal inoculum. Plant lignans in flax seeds and hulls averaged 9.2 and 32.0 nmol mg(-1), respectively. The highest net production of enterodiol at 72 and 96 h of incubation was obtained with flax hulls incubated with faecal microbiota. There was no difference in net production of enterodiol between flax products within the first 24 h of incubation. In general, net production of enterolactone over the 96-h time course was significantly higher for flax products incubated with ruminal than with faecal microbiota. Net production of enterolactone at 72 and 96 h of incubation was greater for flax hulls than flax seeds.. Results of the present experiment suggest that, of the metabolites studied, the main mammalian lignan metabolite produced from flax hulls and seeds by ruminal microbiota is enterolactone while faecal microbiota leads mainly to the net production of enterodiol.. This research will improve the understanding of the metabolic pathway of mammalian lignans in dairy cows, in order to enable targeted manipulation of their quantities in milk.

Topics: 4-Butyrolactone; Animals; Cattle; Chromatography, High Pressure Liquid; Feces; Flax; Lignans; Plant Components, Aerial; Plant Extracts; Rumen; Seeds

Flaxseed lignan secoisolariciresinol diglucoside (SDG) has been reported to prevent and alleviate lifestyle-related diseases including diabetes and hypercholesterolaemic atherosclerosis. This study assesses the effect of SDG on the development of diet-induced obesity in mice and the effect of the SDG metabolite enterodiol (END) on adipogenesis in 3T3-L1 adipocytes. We compared body weight, visceral fat weight, liver fat content, serum parameters, mRNA levels of lipid metabolism-related enzymes and adiponectin in mice fed either a low-fat diet (5 % TAG), high-fat diet (30 % TAG) or high-fat diet containing 0.5 and 1.0 % (w/w) SDG for 4 weeks. Administration of SDG to mice significantly reduced high-fat diet-induced visceral and liver fat accumulation, hyperlipaemia, hypercholesterolaemia, hyperinsulinaemia and hyperleptinaemia. SDG also suppressed sterol regulatory element binding protein 1c mRNA level in the liver and induced increases in the adiponectin mRNA level in the white adipose tissue and carnitine palmitoyltransferase I mRNA level in the skeletal muscle. Differentiated 3T3-L1 adipocytes were treated with 0, 5, 10 and 20 mumol/l END and then assayed for mRNA expression of adipogenesis-related genes and DNA binding activity of PPARgamma to the PPAR response element consensus sequence. END induced adipogenesis-related gene mRNA expression including adiponectin, leptin, glucose transporter 4 and PPARgamma, and induced PPARgamma DNA binding activity in 3T3-L1 adipocytes. In conclusion, SDG induced adiponectin mRNA expression and showed beneficial effects on lipid metabolism in diet-induced obesity in mice. Flaxseed lignans are suggested to regulate adipogenesis-related gene expressions through an increase in PPARgamma DNA binding activity.

Topics: 3T3-L1 Cells; Adipocytes; Adiponectin; Animals; Butylene Glycols; Cholesterol; Dietary Fats; Flax; Glucosides; Insulin; Leptin; Lignans; Male; Mice; Mice, Inbred C57BL; Obesity; PPAR gamma; RNA, Messenger; Triglycerides

Lignans are plant compounds metabolized in the mammalian gut to produce the estrogenic enterolignans, enterodiol (ED) and enterolactone (EL). Because estrogens have been linked to breast cancer etiology, enterolignans could affect breast cancer risk, but to our knowledge, the mechanisms by which they exert their estrogenic and/or anti-estrogenic effects in humans are still unclear. To better understand how estrogenic compounds from the food, such as the enterolignans, might influence breast cancer progression and their mechanisms to interfere with human estrogen receptor (ER) signalling in hormone-dependant diseases, we examined and compared the ability of ED, EL and 17beta-estradiol (E2) to induce the transactivation of ERalpha and ERbeta, to modulate ERalpha target genes, to exert either growth stimulatory or anti-proliferative effects and finally to modulate MCF-7 cell migration by acting on matrix metalloproteases (MMP)-2 and -9, at concentrations that are achievable through a lignan-rich diet. This study indicates that enterolignans show distinct properties for transactivation of ERalpha and ERbeta. ED, as E2, induces ERalpha transcriptional activation through transactivation functions AF-1 and AF-2, while EL is less efficient in inducing AF-1, acting predominantly through AF-2. Furthermore, ED and EL modulate ERalpha mRNA and protein contents as well as MCF-7 cell proliferation and secreted MMP activities in a different way. Enterolignans are compounds of wide interest nowadays and our results help to unveil their mechanisms of action on ER, emphasizing the fact that the dietary load in lignans could be of importance in the balance between being risk or chemopreventive factors for breast cancer and women's health.

Topics: 4-Butyrolactone; Animals; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; CHO Cells; Cricetinae; Cricetulus; Estrogen Receptor alpha; Flavonoids; HeLa Cells; Humans; Lignans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Phenols; Phytoestrogens; Polyphenols; Reverse Transcriptase Polymerase Chain Reaction; Transcriptional Activation

Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis, which is crucial in cancer progression. We have previously shown that estradiol (E2) increases VEGF in breast cancer. Phytoestrogens are potential compounds in breast cancer prevention and treatment by poorly understood mechanisms. The main phytoestrogens in Western diet are lignans, and flaxseed is a rich source of the mammalian lignans enterodiol and enterolactone.. In the present study, ovariectomized mice were treated with continuous release of E2. MCF-7 tumors were established and mice were fed with basal diet or 10% flaxseed, and two groups that were fed basal diet received daily injections with enterodiol or enterolactone (15 mg/kg body weight).. We show that flaxseed, enterodiol, and enterolactone counteracted E2-induced growth and angiogenesis in solid tumors. Extracellular VEGF in vivo, sampled using microdialysis, in all intervention groups was significantly decreased compared with tumors in the basal diet group. Our in vivo findings were confirmed in vitro. By adding enterodiol or enterolactone, E2-induced VEGF secretion in MCF-7 cells decreased significantly without agonistic effects. The increased VEGF secretion by E2 in MCF-7 cells increased the expression of VEGF receptor-2 in umbilical vein endothelial cells, suggesting a proangiogenic effect by E2 by two different mechanisms, both of which were inhibited by the addition of lignans.. Our results suggest that flaxseed and its lignans have potent antiestrogenic effects on estrogen receptor-positive breast cancer and may prove to be beneficial in breast cancer prevention strategies in the future.

Topics: 4-Butyrolactone; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Endothelium, Vascular; Estradiol; Female; Flax; Humans; Lignans; Mice; Neoplasm Transplantation; Neovascularization, Pathologic; Phytoestrogens; Umbilical Veins; Vascular Endothelial Growth Factor A

Enterolignans are phytoestrogenic compounds derived from the conversion of dietary lignans by the intestinal microflora that may be protective against cardiovascular diseases and cancer. To evaluate the use of enterolignans as biomarkers of dietary lignan intake, we studied the relation between plasma and dietary lignans. We determined the dietary intake of 4 lignans (secoisolariciresinol (SECO), matairesinol (MAT), pinoresinol, and lariciresinol) using the European Prospective Investigation into Cancer and Nutrition FFQ, and plasma enterodiol (END) and enterolactone (ENL) concentrations were determined by liquid chromatography-tandem mass spectrometry. The population consisted of 637 men and women, aged 19-75 y, participating in a case-control study on colorectal adenomas. Participants did not use antibiotics in the preceding calendar year. We found a modest association between lignan intake and plasma END (Spearman r = 0.09, P = 0.03) and ENL (Spearman r = 0.18, P <0.001). The correlation of total lignan intake with plasma enterolignans was slightly stronger than that of only SECO plus MAT. The plasma concentrations of both END and ENL were associated with intake of dietary fiber and vegetable protein but not with intake of other macronutrients. The relation between lignan intake and plasma END was modulated by age and previous use of antibiotics, whereas for ENL, it was modulated by weight, current smoking, and frequency of defecation. However, even when we included these nondietary factors in the regression models, the explained variance in plasma END and ENL remained low (2 and 13%, respectively).

Topics: 4-Butyrolactone; Adenomatous Polyps; Adult; Aged; Case-Control Studies; Colonoscopy; Colorectal Neoplasms; Diet; Female; Humans; Lignans; Male; Middle Aged; Phytoestrogens

The flaxseed lignan secoisolariciresinol diglucoside (SDG) and mammalian lignans enterodiol (ED) and enterolactone (EL) were previously shown to be effective antioxidants against DNA damage and lipid peroxidation. Others reported inhibition of activated cell chemiluminescence by supra-physiological concentrations of secoisolariciresinol (SECO), ED and EL. Thus, we evaluated the antioxidant efficacy of potential physiological concentrations of SDG, SECO, ED and EL against 1,1-diphenyl-2-picrylhydrazyl (DPPH()), and 2,2'-azo-bis(2-amidinopropane) dihydrochloride (AAPH)-initiated peroxyl radical plasmid DNA damage and phosphatidylcholine liposome lipid peroxidation. SDG and SECO were effective (p<0.01) antioxidants against DPPH() at 25-200muM; whereas, ED and EL were inactive. Efficacy of lignans and controls against AAPH peroxyl radical-induced DNA damage was: SDG>SECO=17alpha-estradiol>ED=EL>genistein>daidzein. Lignan efficacy against AAPH-induced liposome lipid peroxidation was: SDG>SECO=ED=EL. Plant lignan antioxidant activity was attributed to the 3-methoxy-4-hydroxyl substituents of SDG and SECO, versus the meta mono-phenol structures of ED and EL. Benzylic hydrogen abstraction and potential resonance stabilization of phenoxyl radicals in an aqueous environment likely contributed to the antioxidant activity of the mammalian lignans. These represent likely extra- and intracellular antioxidant activities of flax-derived lignans at concentrations potentially achievable in vivo.

Topics: 4-Butyrolactone; Animals; Biphenyl Compounds; Butylene Glycols; Flax; Free Radical Scavengers; Glucosides; Hydrazines; Lignans; Liposomes; Mammals; Molecular Structure; Picrates; Seeds

During the course of experiments on the transformation of lignans to phytoestrogenic substances, such as enterodiol (END) and enterolactone (ENL), a previously isolated bacterium, Eubacterium (E.) sp. strain SDG-2, capable of phenolic p-dehydroxylation in the biotransformation of secoisolariciresinol diglucoside to END and ENL, was concluded to be Eggerthella (Eg.) lenta (Eg. sp. SDG-2) on the basis of 16S rRNA gene sequence analysis. The bacterium could transform (+)-dihydroxyenterodiol (DHEND, 3a) to (+)-END (1a), but not for (-)-DHEND (3b) to (-)-END (1b) under anaerobic conditions. By incubation of a mixture of (+)- and (-)-dihydroxyenterolactone (DHENL, 4a and 4b) with Eg. sp. SDG-2, only (-)-DHENL (4b) was converted to (-)-ENL (2b), selectively. On the other hand, we isolated a different bacterium, strain ARC-1, capable of dehydroxylating (-)-DHEND (3b) to (-)-END (1b) from human feces. Strain ARC-1 could transform not only (-)-DHEND (3b) to (-)-END (1b), but also (+)-DHENL (4a) to (+)-ENL (2b). However, the bacterium could not transform (+)-DHEND (3a) and (-)-DHENL (4b). Both bacterial strains demonstrated different enantioselective dehydroxylation.

Topics: 4-Butyrolactone; Bacteria, Anaerobic; Biotransformation; Butylene Glycols; Eubacterium; Feces; Glucosides; Humans; Hydroxylation; Intestines; Lignans; Molecular Structure; Phylogeny; RNA, Bacterial; RNA, Ribosomal, 16S; Stereoisomerism

In the course of our experiments on the metabolic conversion of lignans to the estrogenic substances enterodiol (END) and enterolactone (ENL) by human intestinal flora, we isolated two anaerobes, Ruminococcus sp. END-1 and strain END-2, capable of oxidizing END. The former selectively converted (-)-END to (-)-ENL, while the latter selectively converted (+)-END to (+)-ENL, indicating enantioselective oxidation by intestinal bacteria.

Topics: 4-Butyrolactone; Chromatography, High Pressure Liquid; Chromatography, Liquid; Genes, Bacterial; Humans; Intestines; Lignans; Mass Spectrometry; Molecular Structure; Oxidation-Reduction; Phylogeny; RNA, Bacterial; RNA, Ribosomal, 16S; Ruminococcus; Sequence Analysis, RNA; Spectrophotometry, Ultraviolet; Stereoisomerism; Substrate Specificity

This study determined the effect of the mammalian lignans enterolactone (ENL) and enterodiol (END) alone and in combination with the isoflavone genistein (GEN) on the growth of MCF-7 tumors in ovariectomized nude mice. Ovariectomized athymic nude mice with established MCF-7 tumors were fed a basal diet (AIN-93G) and divided into 5 groups that received daily subcutaneous injections (10 mg/kg body weight (BW)) of ENL, END, GEN, a mixture of these compounds (MIX), or vehicle as a negative control for 22 weeks. A positive control group was implanted with an estradiol pellet in order to establish an estrogenic tumor growth response. In the ENL- and END-treated mice, palpable tumors regressed significantly by 91 and 83%, respectively, resulting in final tumors that were similar to the negative control tumors. However, tumor cell apoptosis was significantly enhanced by the lignans. In the GEN-treated mice, tumors initially regressed significantly by 64% but regression ceased following prolonged treatment, resulting in final tumors that were significantly larger compared to negative control, ENL-, and END-treated mice, in part by increasing tumor cell proliferation. The MIX treatment significantly regressed palpable tumors by 87% similar to negative control group, with no effects on tumor cell apoptosis or proliferation. The isoflavone GEN alone promoted the growth of established MCF-7 human breast cancer xenografts after prolonged treatment while the mammalian lignans ENL and END did not. When these phytoestrogens were given in combination, no tumor growth-promoting effects were observed. (c) 2005 Wiley-Liss, Inc.

Topics: 4-Butyrolactone; Animals; Apoptosis; Body Weight; Cell Line, Tumor; Cell Proliferation; Drug Therapy, Combination; Feeding Behavior; Genistein; Humans; Lignans; Mice; Mice, Nude; Neoplasms; Ovariectomy; Xenograft Model Antitumor Assays

Phytoestrogens are plant compounds that have been proposed to have a variety of health benefits. The aim of this study was to assess the effects of these compounds on a number of physiological endpoints. Subjects were given a single intake of a phytoestrogen-rich (80 mg total phytoestrogens) supplement containing soy, rye and linseed (Phase 1), followed by a week-long intervention using the same supplement (Phase 2) (80 mg total phytoestrogens daily). A number of biochemical endpoints were assessed including urinary phytoestrogen metabolites, lipids, antioxidant status, DNA damage and insulin-like growth factor-1 (IGF-1) and IGF binding protein-1 (IGFBP-1) and -3 (IGFBP-3). Ten healthy female subjects took part in the study. Excretion of the isoflavones genistein, daidzein and equol in urine increased in both phases of the study. No other endpoint was altered in Phase 1. However, in Phase 2, concentrations of IGF-1 and IGFBP-3 were increased by phytoestrogen supplementation [IGF-1, median (IQ range), baseline 155 (123, 258), postweek 265 (228, 360) ng/ml, P<.05; IGFBP-3, baseline 3725 (3631, 4196), postweek 4420 (4192, 4935) ng/ml, P<.05]. There was no effect of supplementation on lipids or markers of antioxidant status. Short-term phytoestrogen supplementation increases urinary phytoestrogen excretion and increases IGF-1 and IGFBP-3. These results require elucidation in further controlled studies.

Topics: Adult; Antioxidants; Diet; DNA Damage; Equol; Female; Flax; Genistein; Glycine max; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Isoflavones; Kinetics; Lignans; Lipids; Middle Aged; Phytoestrogens

The human intestinal microbiota is essential for the conversion of the dietary lignan secoisolariciresinol diglucoside (SDG) via secoisolariciresinol (SECO) to the enterolignans enterodiol (ED) and enterolactone (EL). However, knowledge of the species that catalyse the underlying reactions is scant. Therefore, we focused our attention on the identification of intestinal bacteria involved in the conversion of SDG. Strains of Bacteroides distasonis, Bacteroides fragilis, Bacteroides ovatus and Clostridium cocleatum, as well as the newly isolated strain Clostridium sp. SDG-Mt85-3Db, deglycosylated SDG. Demethylation of SECO was catalysed by strains of Butyribacterium methylotrophicum, Eubacterium callanderi, Eubacterium limosum and Peptostreptococcus productus. Dehydroxylation of SECO was catalysed by strains of Clostridium scindens and Eggerthella lenta. Finally, the newly isolated strain ED-Mt61/PYG-s6 catalysed the dehydrogenation of ED to EL. The results indicate that the activation of SDG involves phylogenetically diverse bacteria, most of which are members of the dominant human intestinal microbiota.

Topics: 4-Butyrolactone; Bacteria, Anaerobic; Butylene Glycols; Culture Media; Glucosides; Humans; Intestines; Lignans; Phylogeny

The scientific debate on the role of dietary phytoestrogens for prevention of breast cancer is still ongoing. We previously reported an inverse association between dietary phytoestrogen intake and premenopausal breast cancer risk and now examine the relationship with plasma phytoestrogen concentrations.. We measured enterolactone (mammalian lignan) and genistein (isoflavone) concentrations in plasma samples of 220 premenopausal cases and 237 age-matched controls from a population-based case-control study in Germany.. Median plasma enterolactone concentrations in cases and controls were 6.3 and 9.7 nmol/l, respectively, and median genistein concentrations were 4.5 and 3.7 nmol/l, respectively. Premenopausal breast cancer risk decreased with increasing plasma enterolactone concentrations. Adjusted odds ratios (95% confidence intervals) were 0.42 (0.20-0.90) and 0.38 (0.17-0.85) (P for trend 0.007) for women in the third and fourth quartile of plasma enterolactone compared to those in the lowest quartile. There was no significant association between plasma genistein concentration and premenopausal breast cancer risk.. Using biomarkers of phytoestrogen intake, we confirmed the strong inverse association between enterolactone and premenopausal breast cancer risk as found with dietary intake estimates. This result gives support to the potential role of mammalian lignans for breast cancer prevention among premenopausal women in Western populations.

Topics: 4-Butyrolactone; Adult; Breast Neoplasms; Case-Control Studies; Diet; Female; Genistein; Humans; Lignans; Middle Aged; Phytoestrogens; Plant Preparations; Premenopause; Risk Factors

Lignans are biphenolic compounds that occur in foods of plant origin such as whole grains, seeds, fruits and vegetables, and beverages, such as coffee and tea. Plant lignans are converted by intestinal bacteria into the enterolignans, enterodiol and enterolactone. Enterolignans possess several biological activities, whereby they may influence carcinogenesis. We studied the associations between plasma enterolignans and the risk of colorectal adenomas in a Dutch case-control study. Colorectal adenomas are considered to be precursors of colorectal cancer. Cases (n = 532) with at least one histologically confirmed colorectal adenoma and controls (n = 503) with no history of any type of adenoma were included. Plasma enterodiol and enterolactone concentrations were measured by liquid chromatography with tandem mass spectrometry. Associations were stronger for incident than for prevalent cases. When only incident cases (n = 262) were included, high compared to low plasma concentrations of enterodiol were associated with a reduction in colorectal adenoma risk after adjustment for confounding variables. Enterodiol odds ratios (95% confidence intervals) were 1.00, 0.69 (0.42-1.13), 0.60 (0.37-0.99), and 0.53 (0.32-0.88) with a significant trend (P = 0.01) through the quartiles. Although enterolactone plasma concentrations were 10-fold higher, enterolactone's reduction in risk was not statistically significant (P for trend = 0.09). Use of oral antibiotic therapy could decrease the plasma concentrations of enterolactone. Exclusion of antibiotic users resulted in similar odds ratios for both enterolignans, but the association for enterolactone became somewhat stronger (P = 0.05 versus P = 0.09). We observed a substantial reduction in colorectal adenoma risk among subjects with high plasma concentrations of enterolignans, in particular, enterodiol. These findings could be important in the prevention of colorectal adenomas.

Topics: 4-Butyrolactone; Adenoma; Case-Control Studies; Chromatography, Liquid; Colorectal Neoplasms; Diet; Female; Humans; Incidence; Lignans; Middle Aged; Netherlands; Odds Ratio; Retrospective Studies; Spectrometry, Mass, Electrospray Ionization; Surveys and Questionnaires

The mammalian lignan enterolactone (ENL) produced from plant lignans, e. g. secoisolariciresinol diglycoside (SDG), may protect against various cancers in humans. The present work aims to evaluate the effect of flaxseed on tumour formation in multiple intestinal neoplasia (Min) mice, a model for colon tumorigenesis.. Male and female Min mice were fed either with a non-fibre control diet or the same diet supplemented with 0.5 % (w/w) defatted flaxseed meal. Conversion of SDG to the mammalian lignans enterodiol (END) and ENL in the gut, and plasma ENL, were measured by HPLC with coulometric electrode array detector (CEAD) and timeresolved fluoroimmunoassay, respectively. Wild-type mice were also fed with the experimental diets in order to see whether lignan metabolism is different in Min and wild-type mice.. The total number of adenomas or their size in the small intestine was not different in the flaxseed and control groups. The flaxseed group had a tendency for a decreased number of colon adenomas in both genders. Gender and genotype based differences were found in the intestinal ENL levels. When compared to Min females, Min males in the flaxseed group had several fold higher ENL levels in the small intestine (Min males 125 +/- 124.5 nmol/g vs. females 22.8 +/- 16.0 nmol/g, P = 0.048) and caecum (47.6 +/- 31.6 nmol/g vs. females 14.5 +/- 6.6 nmol/g, P = 0.001). Presence of adenomas in the gut influences the intestinal lignan metabolism. Min mice had less intestinal END and ENL as compared with the wild-type mice (P < 0.05). Mean plasma ENL increased 7-fold during the flaxseed feeding (7 nmol/L in control vs. 50 nmol/L in flaxseed group) but no differences between gender and genotype were found. The plasma ENL level did not correlate with adenoma number in the small intestine and colon.. The number of intestinal adenomas in the Min mouse model is not related to ENL level in plasma nor is it associated with the levels of intestinal lignans. A gender difference in ENL lignan metabolism was found in the gut but not in the plasma.

Topics: Adenoma; Animals; Chromatography, High Pressure Liquid; Disease Models, Animal; Female; Flax; Fluoroimmunoassay; Genotype; Intestinal Neoplasms; Lignans; Male; Mice; Mice, Mutant Strains; Neoplasms, Multiple Primary; Phytoestrogens; Random Allocation; Sex Factors

The enterolignans enterolactone and enterodiol are phytoestrogens that are formed from plant lignans by microorganisms in the human colon. Enterolignans circulate in plasma as conjugates. We hypothesized that conjugation of enterolignans takes place in colon epithelial cells, and studied the time course of uptake and metabolism of enterolactone and enterodiol in three human colon epithelial cell lines. In addition, the conjugates were identified by mass spectrometry with accurate mass measurement (LC/QTOFMS/MS). Intracellular levels of conjugated enterolactone and enterodiol in HT29 cells rose immediately after starting the exposure. This was accompanied by a rapid decrease in free enterolactone and enterodiol in the exposure medium of HT29 and (un)differentiated CaCo-2 but not of CCD841CoTr cells. Conjugation and excretion of enterolactone and enterodiol was complete within 8 h, except for enterodiol in CaCo-2 cells ( approximately 48 h). Enterolactone appears to be more rapidly metabolized and/or excreted than enterodiol, and also the appearance of conjugated enterolactone in medium is less affected by the presence of enterodiol than vice versa. Total (free plus conjugated) enterolignan concentrations remained constant throughout the experiments. Three conjugates were identified in exposure medium of HT29 cells: enterolactone-sulfate, enterolactone-glucuronide, and enterodiol-glucuronide. Taken together, our data suggest that phase II metabolism of enterolactone and enterodiol already may take place during uptake in the colon and that colon epithelial cells may be responsible for this metabolism.

Topics: 4-Butyrolactone; Caco-2 Cells; Cell Line; Colon; HT29 Cells; Humans; Intestinal Mucosa; Lignans; Metabolic Clearance Rate

High concentrations of enterolignans in plasma are associated with a lower risk of acute coronary events. However, little is known about the absorption and excretion of enterolignans. The pharmacokinetic parameters and urinary excretion of enterodiol and enterolactone were evaluated after consumption of their purified plant precursor, secoisolariciresinol diglucoside (SDG). Twelve healthy volunteers ingested a single dose of purified SDG (1.31 micromol/kg body wt). Enterolignans appeared in plasma 8-10 h after ingestion of the purified SDG. Enterodiol reached its maximum plasma concentration 14.8 +/- 5.1 h (mean +/- SD) after ingestion of SDG, whereas enterolactone reached its maximum 19.7 +/- 6.2 h after ingestion. The mean elimination half-life of enterodiol (4.4 +/- 1.3 h) was shorter than that of enterolactone (12.6 +/- 5.6 h). The mean area under the curve of enterolactone (1762 +/- 1117 nmol/L . h) was twice as large as that of enterodiol (966 +/- 639 nmol/L . h). The mean residence time for enterodiol was 20.6 +/- 5.9 h and that for enterolactone was 35.8 +/- 10.6 h. Within 3 d, up to 40% of the ingested SDG was excreted as enterolignans via urine, with the majority (58%) as enterolactone. In conclusion, a substantial part of enterolignans becomes available in the blood circulation and is subsequently excreted. The measured mean residence times and elimination half-lives indicate that enterolignans accumulate in plasma when consumed 2-3 times a day and reach steady state. Therefore, plasma enterolignan concentrations are expected to be good biomarkers of dietary lignan exposure and can be used to evaluate the effects of lignans.

Topics: 4-Butyrolactone; Adult; Blood Specimen Collection; Butylene Glycols; Dietary Supplements; Female; Glucosides; Humans; Lignans; Male; Reference Values

Estrogen plays a major role in breast cancer development and progression. Breast tissue and cell lines contain the necessary enzymes for estrogen synthesis, including aromatase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD). These enzymes can influence tissue exposure to estrogen and therefore have become targets for breast cancer treatment and prevention. This study determined whether the isoflavone genistein (GEN) and the mammalian lignans enterolactone (EL) and enterodiol (ED) would inhibit the activity of aromatase and 17beta-HSD type 1 in MCF-7 cancer cells, thereby decreasing the amount of estradiol (E2) produced and consequently cell proliferation. Results showed that 10 microM EL, ED and GEN significantly decreased the amount of estrone (E1) produced via the aromatase pathway by 37%, 81% and 70%, respectively. Regarding 17beta-HSD type 1, 50 microM EL and GEN maximally inhibited E2 production by 84% and 59%, respectively. The reduction in E1 and E2 production by EL and the reduction in E2 production by GEN were significantly related to a reduction in MCF-7 cell proliferation. 4-Hydroxyandrostene-3,17-dione (50 microM) did not inhibit aromatase but inhibited the conversion of E1 to E2 by 78%, suggesting that it is a 17beta-HSD type 1 inhibitor. In conclusion, modulation of local E2 synthesis is one potential mechanism through which ED, EL and GEN may protect against breast cancer.

Topics: 17-Hydroxysteroid Dehydrogenases; 4-Butyrolactone; Animals; Aromatase; Aromatase Inhibitors; Breast Neoplasms; Cell Line, Tumor; Estradiol; Female; Genistein; Humans; Lignans; Mammals; Phytoestrogens

Enterolactone and enterodiol are phytoestrogens with structural similarity to endogenous estrogens. Because of their biological activities, they may affect the development of several diseases. To quantify enterodiol and enterolactone in plasma, we developed and validated a liquid chromatography-tandem mass spectrometry method with electrospray ionization using 13C3 labeled isotopes. The method consists of a simple enzymatic hydrolysis and ether extraction followed by a rapid LC separation (run-time of 11 min). Detection limits as low as 0.15 nM for enterodiol and 0.55 nM for enterolactone were achieved. The within-run R.S.D. ranges from 3 to 6% and the between-run R.S.D. ranges from 10 to 14% for both enterolignans. This method allows simple, rapid, and sensitive quantification, and is suitable for measuring large numbers of samples.

Topics: 4-Butyrolactone; Adult; Carbon Isotopes; Chromatography, Liquid; Drug Stability; Female; Humans; Lignans; Male; Mass Spectrometry; Middle Aged; Reproducibility of Results

Lignans are dietary diphenolic compounds which require activation by intestinal bacteria to exert possible beneficial health effects. The intestinal ecosystem plays a crucial role in lignan metabolism, but the organisms involved are poorly described. To characterize the bacterial communities responsible for secoisolariciresinol (SECO) activation, i.e., the communities that produce the enterolignans enterodiol (ED) and enterolactone (EL), a study with 24 human subjects was undertaken. SECO activation was detected in all tested fecal samples. The intestinal bacteria involved in ED production were part of the dominant microbiota (6 x 10(8) CFU g(-1)), as revealed by most-probable-number enumerations. Conversely, organisms that catalyzed the formation of EL occurred at a mean concentration of approximately 3 x 10(5) CFU g(-1). Women tended to have higher concentrations of both ED- and EL-producing organisms than men. Significantly larger amounts of EL were produced by fecal dilutions from individuals with moderate to high concentrations of EL-producing bacteria. Two organisms able to demethylate and dehydroxylate SECO were isolated from human feces. Based on 16S rRNA gene sequence analyses, they were named Peptostreptococcus productus SECO-Mt75m3 and Eggerthella lenta SECO-Mt75m2. A new 16S rRNA-targeted oligonucleotide probe specific for P. productus and related species was designed and further used in fluorescent in situ hybridization experiments, along with five additional group-specific probes. Significantly higher proportions of P. productus and related species (P = 0.012), as well as bacteria belonging to the Atopobium group (P = 0.035), were typical of individuals with moderate to high concentrations of EL-producing communities.

Topics: 4-Butyrolactone; Actinobacteria; Adult; Bacteria, Anaerobic; Butylene Glycols; Colony Count, Microbial; Culture Media; Female; Flow Cytometry; Glucosides; Humans; In Situ Hybridization, Fluorescence; Intestines; Lignans; Male; Middle Aged; Peptostreptococcus; Phytoestrogens

The mammalian lignans enterolactone (ENL) and enterodiol, commonly found in human plasma and urine, are phytoestrogens that may contribute to the prevention of breast cancer and coronary heart disease. They are formed by the conversion of dietary precursors such as secoisolariciresinol and matairesinol lignans by the colonic microflora. The identification of lignins, cell-wall polymers structurally related to lignans, as precursors of mammalian lignans is reported here for the first time. In study 1, rats were fed rye or wheat bran (15% diet) for 5 d. Untreated brans and brans extracted with solvents to remove lignans were compared. ENL was estimated in urine samples collected for 24 h by time-resolved fluoroimmunoassay. ENL urinary excretion was reduced from 18.6 to 5.3 nmol/d (n=8; P<0.001) when lignans were removed from rye bran and from 30.5 to 6.2 nmol/d (P<0.001) when they were removed from wheat bran. These results suggest that lignins, embedded in the cell wall and retained in the bran during solvent extraction, account for 26-32% of the ENL formed from cereal brans. In study 2, rats were fed a deuterated synthetic lignin (0.2% diet) together with wheat bran (15%) for 3 d. The detection of deuterated ENL by LC-tandem MS in urine (20 nmol/d) clearly confirms the conversion of lignin into mammalian lignans. More research is warranted to determine the bioavailability of lignins in the human diet.

Topics: 4-Butyrolactone; Animals; Biological Availability; Cell Wall; Cellulases; Chromatography, Liquid; Deuterium; Diet; Gas Chromatography-Mass Spectrometry; Glycoside Hydrolases; Isoflavones; Lignans; Lignin; Male; Phytoestrogens; Plant Preparations; Rats; Rats, Wistar; Secale; Spectrometry, Mass, Electrospray Ionization; Triticum

Quantitative analytical methods for the 17 phytoestrogens containing isoflavonoids, lignans, and mycoestrogens in herbs were developed, and the amount of phytoestrogens was determined in 22 traditional medicinal herbs. The focus of this study was to simplify the purification procedure for removing many kinds of interferences in herbs, and to select adequate derivatization reagent for getting desirable selectivity and sensitivity in the quantitative determination of phytoestrogens. To satisfy these goals, we performed a solid-phase extraction with Oasis HLB cartridges following enzymatic and acidic hydrolysis, and we used the mixture of MSTFA/NH4I/DTE (1000:4:5, v/w/w) to form TMS derivatives of phytoestrogens. Overall recovery was more than 84% in all of the phytoestrogens, and the limit of quantification for phytoestrogens in herbs were set at 0.2 microg/g. Coefficient of variation percentages were in the range of 0.18-15.68% (within-day) and 0.23-16.61% (day-to-day), respectively. Most of the isoflavonoids and lignans were found in all of the herbs, but mycoestrogens were not detected at all. The Leguminosae family proved to be the richest source of isoflavonoids. Lignans such as enterodiol and enterolactone were detected at low concentration in most of the herbs. These results indicate that this assay is accurate and reliable for the determination of phytoestrogens in herbs. Also, information regarding the phytoestrogen contents in traditional medicinal herbs is useful in the prevention and treatment of chronic diseases such as cancer, osteoporosis, dementia, and cardiovascular disease.

Topics: 4-Butyrolactone; Chemistry Techniques, Analytical; Fabaceae; Flavonoids; Gas Chromatography-Mass Spectrometry; Lignans; Phytoestrogens; Plants, Medicinal; Reproducibility of Results; Sensitivity and Specificity

[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.

Topics: 4-Butyrolactone; Furans; Hydrogenation; Lignans; Picea; Stereoisomerism

Phytoestrogens (isoflavones and lignans) are of increasing interest due to their potential to prevent certain types of complex diseases. However, epidemiological evidence is needed on the levels of phytoestrogens and their metabolites in foods and biological fluids in relation to risk of these diseases. We report an assay for phytoestrogens which is sensitive, accurate, and uses low volumes of sample. Suitable for epidemiological studies, the assay consists of a simple sample preparation procedure and has been developed for the analysis of five isoflavones (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone), which requires only 200 microl of urine and utilizes one solid-phase extraction stage for sample preparation prior to derivatization for GC/MS analysis. Limits of detection were in the region 1.2 ng/ml (enterodiol) to 5.3ng/ml (enterolactone) and the method performed well in the UK Government's Food Standards Agency-sponsored quality assurance scheme for phytoestrogens. For the first time, average levels of all the above phytoestrogens were measured in samples of urine collected from a free living population sample of women. Results show a large range in both the amount and the type of phytoestrogens excreted.

Topics: 4-Butyrolactone; Equol; Female; Gas Chromatography-Mass Spectrometry; Genistein; Humans; Isoflavones; Lignans; Molecular Structure; Phytoestrogens; Plant Preparations; Reproducibility of Results; Sensitivity and Specificity

In an international context of promoting scientific research on food safety, the interest in molecules having potential hormonal disrupting effects is growing. While industrial endocrine disruptors (phthalates, alkylphenols, PCBs, etc.) have been studied for several years, natural compounds like phytoestrogens remain less investigated. Accordingly, a research project was initiated with its main objectives to develop efficient analytical methods for a wide range of phytoestrogens in various food matrices, and to evaluate their occurrence in food products. Electrospray ionization with tandem mass spectrometric (MS/MS) analysis of isoflavones (genistein, daidzein, equol, formononetin, biochanin A), lignans (enterolactone, enterodiol), and coumestans (coumestrol) was investigated. This study revealed the formation of a large number of fragment ions in both positive and negative modes, corresponding to specific cleavages of the hydroxyl, carbonyl, and/or methoxy groups, and to Retro-Diels-Alder reactions. An LC/ESI-MS/MS method was developed consistent with the 2002/657/EC European decision criteria. An extraction and clean-up method was developed for milk samples. The identification limit for the proposed method appears to be under 1 ng/mL. The developed methodology was applied to various milk samples, and the occurrence of isoflavones (particularly equol) was demonstrated in the concentration range 1-30 ng/mL. The efficiency of the proposed analytical method permitted evaluation of a new and promising approach to a global risk assessment of natural estrogenic active substances including phytoestrogens and their metabolites.

Topics: 4-Butyrolactone; Animals; Cattle; Chromans; Chromatography, Liquid; Coumestrol; Equol; Estrogens, Non-Steroidal; Genistein; Ions; Isoflavones; Lignans; Milk; Molecular Structure; Phytoestrogens; Plant Preparations; Spectrometry, Mass, Electrospray Ionization

Flaxseed has been shown to reduce the metastasis of estrogen receptor negative (ER-) human breast cancer in nude mice. This study determined whether enterodiol (ED) and enterolactone (EL), metabolites of plant lignans exceptionally rich in flaxseed, and tamoxifen (TAM), alone or in combination, can influence the various steps of metastasis, that is, breast cancer cell adhesion, invasion and migration, of two ER- human breast cancer cell lines, MDA-MB-435 and MDA-MB-231. The inhibition by ED, EL or TAM (1-5 microM) of cell adhesion to Matrigel or extracellular matrices, fibronectin, laminin, and type IV collagen, as well as cell invasion was dose dependent in both cell lines. When ED, EL and TAM were combined at 1 microM, a greater inhibitory effect on cell adhesion and invasion was observed than with either compound alone. ED and EL at doses of 0.1-10 microM reduced cell migration, but TAM had no effect at 0.1 and 1 microM, and exhibited a stimulatory effect at 10 microM. It is concluded that lignans and TAM, alone or in combination, can inhibit the steps involved in the metastasis cascade. Although more investigations are required, the study also suggests that the intake of the lignan-rich flaxseed may not antagonize the effect of TAM in ER- breast cancer cells.

Topics: 4-Butyrolactone; Antineoplastic Agents; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Movement; Female; Humans; Lignans; Neoplasm Invasiveness; Neoplasm Metastasis; Phytotherapy; Receptors, Estrogen; Tamoxifen

Lignans are plant compounds metabolized in the gut to produce the phytoestrogens enterolactone and enterodiol. Reduced breast cancer risks associated with higher urinary lignan excretion may be related to competitive inhibition of endogenous estrogens. Evidence exists that associations with reproductive risk factors for breast cancer differ according to cytochrome P450c17alpha (CYP17) genotype. Genetic variability in estrogen metabolism could affect lignan metabolism thereby modifying risk associations. We examined breast cancer risk, dietary lignans and CYP17 genotype among 207 women with primary, incident, histologically confirmed breast cancer and 188 controls frequency matched to cases by age and county of residence. Self-reported frequency of intake of 170 foods and beverages during the 2 y before the interview and other relevant data were collected by detailed in-person interviews. Dietary lignan intake was expressed as the sum of enterolactone and enterodiol production from foods. Odds ratios (OR) and 95% confidence intervals (CI) were estimated by unconditional logistic regression, adjusting for age, education and other breast cancer risk factors. Women in the highest tertile of dietary lignans tended to have reduced breast cancer risk (OR 0.45, 95% CI 0.20-1.01 and OR 0.59, 95% CI 0.28-1.27, pre- and postmenopausal women, respectively). Substantially reduced risks in the highest tertile of lignans were observed for premenopausal women with at least one A2 allele (OR 0.12, 95% CI 0.03-0.50). Our results suggest that CYP17 genotype may be important in modifying the effect on breast cancer risk of exogenous estrogens, particularly for premenopausal women.

Topics: 4-Butyrolactone; Adult; Aged; Breast Neoplasms; Case-Control Studies; Dose-Response Relationship, Drug; Female; Food Analysis; Genotype; Humans; Interviews as Topic; Lignans; Middle Aged; Odds Ratio; Premenopause; Risk Factors; Steroid 17-alpha-Hydroxylase

Two sensitive competitive-type solid-phase immunoassays for serum daidzein analysis have been developed and optimized. The first is a chemiluminescent enzyme immunoassay that uses black polystyrene microtiter wells in which daidzein-specific antibodies raised in rabbits are immobilized and a daidzein derivative is coupled to horseradish peroxidase (HRP) as a label. The HRP activity of the antibody-bound tracer is measured with an enhanced chemiluminescent system (luminol/H2O2/enhancer). The second immunoassay is based on the use of bovine serum albumin-daidzein derivative immobilized on microtiter plates and a secondary anti-rabbit IgG-Fc fragment conjugated with 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Formation of the complex Eu3+-BCPDA enables time-resolved fluorescence-mode detection of the amount of antibody bound to the immobilized antigen. Both methods fulfilled all the requirements of accuracy and precision. The detection limit was the same for each method, 10 pg/well; this is better than that of other immunoassays. The specificity of the two methods was different, because of their competitive-type mechanisms. The performance of the chemiluminescence method is better, because the cross-reactivity of the main interfering compound (genistein) was 5%, compared with 25% for the time-resolved fluoroimmunoassay.

Topics: 4-Butyrolactone; Cross Reactions; Dose-Response Relationship, Immunologic; Equilin; Female; Fluorescent Dyes; Genistein; Haptens; Horseradish Peroxidase; Humans; Immunoenzyme Techniques; Immunoglobulin Fc Fragments; Isoflavones; Lignans; Luminescent Measurements; Molecular Structure; Phenanthrolines; Polystyrenes; Radioimmunoassay; Serum Albumin, Bovine; Steroids

The metabolism of the plant lignans matairesinol, secoisolariciresinol, pinoresinol, syringaresinol, arctigenin, 7-hydroxymatairesinol, isolariciresinol, and lariciresinol by human fecal microflora was investigated to study their properties as mammalian lignan precursors. The quantitative analyses of lignan precursors and the mammalian lignans enterolactone and enterodiol were performed by HPLC with coulometric electrode array detector. The metabolic products, including mammalian lignans, were characterized as trimethylsilyl derivatives by gas chromatography-mass spectrometry. Matairesinol, secoisolariciresinol, lariciresinol, and pinoresinol were converted to mammalian lignans only. Several metabolites were isolated and tentatively identified as for syringaresinol and arctigenin in addition to the mammalian lignans. Metabolites of 7-hydroxymatairesinol were characterized as enterolactone and 7-hydroxyenterolactone by comparison with authentic reference compounds. A metabolic scheme describing the conversion of the most abundant new mammalian lignan precursors, pinoresinol and lariciresinol, is presented.

Topics: 4-Butyrolactone; Chromatography, High Pressure Liquid; Feces; Gas Chromatography-Mass Spectrometry; Lignans; Plant Extracts

Recent studies have shown that the mammalian lignans enterodiol (END) and enterolactone (ENL) are biotransformed in vitro by hepatic microsomes from rats and humans to various metabolites carrying one additional hydroxy group either at the aromatic or at the aliphatic moiety. To clarify whether these metabolites are also formed in vivo, each lignan was administered intraduodenally at a dose of 10 mg/kg of bw to bile duct-catheterized female Wistar rats and the 6 h bile analyzed by HPLC and GC-MS. With END-dosed rats, three products of aromatic and two of aliphatic monohydroxylation were found, whereas six aromatic and five aliphatic monohydroxylated biliary metabolites were detected after administration of ENL. The metabolites hydroxylated at the aromatic rings were unequivocally identified by comparison with synthetic reference compounds. The structures of the in vivo metabolites arising from aliphatic hydroxylation could not be completely elucidated; they were identical with some of the formerly reported microsomal products according to GC retention times and mass spectra. Significant amounts of most of the metabolites of the mammalian lignans identified in bile were also found in the urine of female rats after oral administration of 10 mg/kg of bw END or ENL and in the urine of female and male Wistar rats after they had been fed a diet containing 5% flaxseed. Thus, the mammalian lignans END and ENL give rise to several hydroxylated metabolites in vivo, which may contribute to the biological effects of these important food constituents.

Topics: 4-Butyrolactone; Animals; Bile; Female; Gas Chromatography-Mass Spectrometry; Lignans; Male; Oxidation-Reduction; Rats; Rats, Wistar

A series of variably substituted derivatives of lignan lactones and diols were prepared using tandem conjugate addition reaction as a key step. These theoretical precursors of the mammalian lignans enterolactone 1 and enterodiol 3 are moderate or weak inhibitors of human aromatase activity.

Topics: 4-Butyrolactone; Aromatase Inhibitors; Catechols; Enzyme Inhibitors; Humans; Lignans; Phenols

Flaxseed has renoprotective effects in animal and human lupus nephritis. We have recently extracted the lignan precursor (secoisolariresinol diglucoside) (SDG) to determine if this more palatable derivative of flaxseed would exert renoprotection similar to the whole flaxseed in the aggressive MRL/lpr lupus mouse model.. 131 MRL/lpr mice were randomly assigned to saline gavage, 600, 1,200 and 4,800 microg lignan gavage groups. At 7 weeks, 6 animals underwent platelet aggregating factor (PAF) lethal challenge and 40 were studied with urine collection to determine the levels of secoisolariresinol, enterodiol and enterolactone in the gavaged animals. A baseline study of 10 saline gavaged animals took place at 6 weeks. 25 animals in the saline gavage, 600 and 1200 microg lignan groups were studied at 14 and 22 weeks for GFR, spleen lymphocyte S-phase and organ weight studies.. Metabolic studies indicated that secoisolariresinol is the major metabolite absorbed and the lowest lignan dose provides a lengthening in survival for the PAF lethal challenge. Body weight, fluid and water intake studies demonstrated that the lignan was well tolerated. Changes in proteinuria, GFR and renal size showed a time- and dose-dependent protection for the lignan precursor. Cervical lymph node size and spleen lymphocyte cells in the S-phase demonstrated modest dose-dependent reductions in the lignan gavaged groups.. SDG was converted in the gut to secoisolariresinol, which was absorbed and well tolerated by the MRL/lpr mice. Renoprotection was evidenced, in a dose-dependent fashion, by a significant delay in the onset of proteinuria with preservation in GFR and renal size. This study suggests that SDG may have a therapeutic role in lupus nephritis.

Topics: 4-Butyrolactone; Animals; Blood Coagulation Factors; Flax; Humans; Lignans; Lupus Nephritis; Mice; Phytotherapy; Platelet Activating Factor; Seeds

Dietary phytoestrogens have a number of biological effects, including endocrine disruption, antioxidant potential, and protein tyrosine kinase inhibition. Secoisolariciresinol, matairesinol, and shonanin are lignan phytoestrogens found in foodstuffs, especially flaxseed. Normally they are glycosidically linked to carbohydrates and in the large intestine are deconjugated from the carbohydrate portion by bacteria. The aglycone lignans can be further modified to form the mammalian phytoestrogens enterodiol, enterolactone, and enterofuran, which are absorbed into the body and excreted in urine. To assess the health implications of phytoestrogens in general populations, knowledge of the quantity in the foods eaten is necessary. This article describes a simple preparative procedure for the assay of secoisolariciresinol, matairesinol, and shonanin in foodstuffs after hydrolytic removal of any conjugated carbohydrate. The difficulties in the practical application of the assay procedure are illustrated and discussed. Analytical results indicating the concentration of secoisolariciresinol, matairesinol, and shonanin in a number of foodstuffs are presented. Also, the mass spectral data of a putative mammalian phytoestrogen, called enterofuran, identified in urine are presented.

Topics: Butylene Glycols; Estrogens, Non-Steroidal; Flax; Food Analysis; Furans; Humans; Isoflavones; Lignans; Mass Spectrometry; Phytoestrogens; Plant Preparations; Reference Standards; Triticum

Seven metabolites were isolated after anaerobic incubation of secoisolariciresinol diglucoside (1) with a human fecal suspension. They were identified as (-)-secoisolariciresinol (2), 3-demethyl-(-)-secoisolariciresinol (3), 2-(3-hydroxybenzyl)-3-(4-hydroxy-3-methoxybenzyl)butane-1,4-diol (4), didemethylsecoisolariciresinol (5), 2(3-hydroxybenzyl)-3-(3,4-dihydroxybenzyl)butane-1,4-diol (6), enterodiol (7) and enterolactone (8). Furthermore, two bacterial strains, Peptostreptococcus sp. SDG-1 and Eubacterium sp. SDG-2, responsible for the transformation of 1 to a mammalian lignan 7, were isolated from a human fecal suspension. The former transformed 2 to 3 and 5, as well as 4 to 6, and the latter transformed 5 to 6 and 7.

Topics: 4-Butyrolactone; Animals; Butylene Glycols; Culture Media; Estrogens; Eubacterium; Feces; Glucosides; Humans; Intestines; Lignans; Magnetic Resonance Spectroscopy; Male; Mass Spectrometry; Peptostreptococcus; Rats; Rats, Wistar

Hepatic microsomes from aroclor-treated male Wistar rats biotransform enterolactone to 12 metabolites, six of which carry an additional hydroxy group at the aromatic and six at the aliphatic moiety according to HPLC/MS and GC/MS analysis. The aromatic hydroxylation products were identified with the help of synthesized reference compounds as enterolactone monohydroxylated in the para position and in both ortho positions of the original phenolic hydroxy group of either aromatic ring. The synthesis of the reference compounds and their spectroscopic characterization is described. Enterodiol is metabolized by hepatic microsomes from aroclor-treated male rats to three aromatic and four aliphatic monohydroxylated metabolites. Aromatic hydroxylation occurs in the para position and the two ortho positions of the original phenolic hydroxy group. Most of the metabolites of enterolactone and enterodiol were also formed with microsomes from uninduced rat, pig, and human liver, suggesting that oxidative metabolism is a common feature in the disposition of these lignans in the mammalian organism.

Topics: 4-Butyrolactone; Animals; Biotransformation; Chlorodiphenyl (54% Chlorine); Chromatography, High Pressure Liquid; Estrogens; Gas Chromatography-Mass Spectrometry; Humans; Hydroxylation; Lignans; Male; Mass Spectrometry; Microsomes, Liver; Oxidation-Reduction; Rats; Rats, Wistar; Swine

The antioxidant activities of the flaxseed lignan secoisolariciresinol diglycoside (SDG) and its mammalian lignan metabolites, enterodiol (ED) and enterolactone (EL), were evaluated in both lipid and aqueous in vitro model systems. All three lignans significantly (p < or = 0.05) inhibited the linoleic acid peroxidation at both 10 and 100 microM over a 24-48 h of incubation at 40 degrees C. In a deoxyribose assay, which evaluates the non site-specific and site-specific Fenton reactant-induced *OH scavenging activity, SDG demonstrated the weakest activity compared to ED and EL at both 10 and 100 microM; the greatest *OH scavenging for ED and EL was observed at 100 microM in both assays. The incubation of pBR322 plasmid DNA with Fenton reagents together with SDG, ED or EL showed that the inhibition of DNA scissions was concentration dependent. The greatest non site-specific activity of lignans was at 100 microM, thus, confirming the results of the deoxyribose test. In contrast, the protective effect of SDG and EL in the site-specific assay was lost and that of ED was minimal. Therefore, the results indicate a structure-activity difference among the three lignans with respect to specific antioxidant efficacy. All three lignans did not exhibit reducing activity compared to ascorbic acid, therefore, did not possess indirect prooxidant activity related to potential changes in redox state of transition metals. The efficacy of SDG and particularly the mammalian lignans ED and EL to act as antioxidants in lipid and aqueous in vitro model systems, at relatively low concentrations (i.e. 100 microM), potentially achievable in vivo, is an evidence of a potential anticarcinogenic mechanism of flaxseed lignan SDG and its mammalian metabolites ED and EL.

Topics: 4-Butyrolactone; Animals; Antioxidants; Butylene Glycols; Emulsions; Estrogens; Flax; Free Radical Scavengers; Glucosides; Hydroxides; Lignans; Linoleic Acid; Lipid Peroxidation; Mammals; Oxidation-Reduction; Seeds

The mammalian lignans enterolactone (ENL) and enterodiol (END) are formed by intestinal bacteria from the plant lignans matairesinol (MAT) and secoisolariciersinol (SEC), respectively, which are ingested with different types of food. ENL and END are weak estrogens. According to epidemiological and biochemical studies, lignans may act as anticarcinogens, but little is known about their genotoxic potential. We have therefore investigated the effects of ENL, END, MAT and SEC on cell-free microtubule assembly and at the following genetic endpoints in cultured male Chinese hamster V79 cells: disruption of the cytoplasmic microtubule complex, induction of mitotic arrest, induction of micronuclei and their characterization by CREST staining, and mutagenicity at the HPRT gene locus. The lignans were tested at concentrations of 200 microM in the cell-free system and 100 microM in cultured cells, which represents the limit of solubility in each assay. The established aneuploidogen diethylstilbestrol and the clastogen 4-nitroquinoline-N-oxide were used as positive reference compounds. As none of the four lignans had any activity at the endpoints studied, we conclude that ENL, END, MAT and SEC are devoid of aneuploidogenic and clastogenic potential under the experimental conditions used in this study.

Topics: 4-Butyrolactone; Aneuploidy; Animals; Bacteria; Butylene Glycols; Cattle; Cell Line; Cell-Free System; Cricetinae; Estrogens; Furans; Hypoxanthine Phosphoribosyltransferase; Intestinal Mucosa; Intestines; Lignans; Male; Micronucleus Tests; Microtubules; Mitosis; Mutagenicity Tests; Mutagens

We examined the distribution and long-term reliability of serum measurements of the two main human lignans, enterolactone and enterodiol, and the isoflavonoid phytoestrogens daidzein, genistein, equol, and O-Desmethylangolensin in the New York University Women's Health Study, a prospective cohort study of sex hormones and breast cancer. Serum samples collected at three yearly visits in 30 premenopausal and 30 postmenopausal women who had not been diagnosed with cancer or cardiovascular disease were included in the study. Assays were carried out by ion-exchange chromatography and capillary gas chromatography-mass spectrometry. Levels of isoflavonoid phytoestrogens were low, often at or below the sensitivity level of the assay. The reliability coefficients for these compounds were also low (< or =0.30). The median levels of enterodiol and enterolactone were 1.52 nmol/liter and 20.2 nmol/liter, respectively, and were comparable with the levels observed in omnivorous Finnish women living in the Helsinki area. A substantial number of women, though, had fairly high levels: for instance, 15% of the assays showed levels of enterolactone greater than the mean level observed in vegetarian Finnish women, i.e., 89.1 nmol/liter (H. Adlercreutz et al., Cancer Detec. Prev., 18: 259-271, 1994). The reliability coefficient of a single measurement of enterolactone was moderately high (0.55), suggesting that serum measurements of this compound could be a useful tool in prospective epidemiological studies with access to repeated blood or serum specimens. For instance, the reliability coefficient of the average of three measurements of enterolactone would be 0.79, a level considered acceptable in light of the other sources of error that are present in epidemiological studies (W. Willett, Stat. Med., 8: 1031-1040, 1989).

Topics: 4-Butyrolactone; Adult; Age Factors; Aged; Bias; Breast Neoplasms; Chromans; Equol; Feasibility Studies; Female; Genistein; Humans; Isoflavones; Lignans; Middle Aged; New York City; Pilot Projects; Postmenopause; Premenopause; Reference Values; Reproducibility of Results; Sensitivity and Specificity; Time Factors

Chinese men have lower incidences of prostate cancer compared to men from Europe and North America. Asians consume large quantities of soya, a rich source of isoflavanoids phyto-oestrogens and have high plasma and urinary levels of these compounds. The mammalian lignans, enterolactone and enterodiol, are another group of weak plant oestrogens and are derived from seeds, cereals and grains. Vegetarians have high plasma and urinary concentrations of lignans.. The concentrations lignans and isoflavonic phyto-oestrogens were determined by gas chromatography-mass spectrometry (GC-MS) in plasma and prostatic fluid from Portuguese, Chinese and British men consuming their traditional diets.. In prostatic fluid the mean concentrations of enterolactone were 31, 162 and 20.3 ng/ml for Hong Kong, Portugal and Britain respectively. Very high levels of enterolactone (> 600 ng/ml) were observed in the prostatic fluid of some of the men from Portugal. High concentrations of equol (3270 ng/ml) and daidzein (532 ng/ml) were found in a sample of prostatic fluid from Hong Kong. Higher mean levels of daidzein were observed in prostatic fluid from Hong Kong at 70 ng/ml, compared to 4.6 and 11.3 ng/ml in samples from Portugal and Britain respectively. Mean levels of daidzein were higher in the plasma samples from Hong Kong (31.3 ng/ml) compared to those from Portugal (1.3 ng/ml) and Britain (8.2 ng/ml). In general, the mean plasma concentrations of enterolactone from the three centres were similar, at 6.2, 3.9 and 3.9 ng/ml in samples from Hong Kong Portugal and Britain respectively.. Higher concentrations of the isoflavanoid phyto-oestrogens, daidzein and equol, were found in the plasma and prostatic fluid of men from Hong Kong compared to those from Britain and Portugal. However, the levels of the lignan, enterolactone, were very much higher in prostatic fluid of Portuguese men. Isoflavanoids and lignans have many interesting properties and may, in part, be responsible for lower incidences of prostate cancer in men from Asia and also some Mediterranean countries. The isoflavanoids from soya, which are present in high concentrations in the prostatic fluid of Asian men, may be protective against prostate disease.

Topics: 4-Butyrolactone; Adult; Aged; Chromans; Equol; Gas Chromatography-Mass Spectrometry; Hong Kong; Humans; Isoflavones; Lignans; Male; Middle Aged; Portugal; Prostate; Semen; United Kingdom

The interactions of human Sex steroid binding protein (SBP) and the lignans [Nordihydrogaiaretic acid (NDGA) enterolactone (Ent), enterodiol (End)] and isoflavonoid phytoestrogens [Equol (Eq), diazein Dad), genistein (Gen)] were studied. The phytoestrogens had different dose-dependent inhibitory effects on steroid binding by SBP. Their relative efficiencies were: Ent> or = NDGA = Eq > Gen for displacing E2 and Eq > Ent > NDGA > Gen for displacing T. End and Dad were much less active. Scatchard analysis suggested that NDGA had similar non- competitive effects on T and E2 binding by reducing the number of binding sites without changing the association constants. But Eq seemed to inhibit E2 binding non-competitively and T binding competitively. NDGA binding to SBP reduced the immunorecognition of SBP by monospecific anti-SBP antibodies, suggesting that NDGA changed SBP immunoreactivity. Unlike NDGA, Eq binding to SBP caused no immunological changes in SBP, indicating qualitative differences in the effects of the lignan and isoflavonoid. Our results indicate that phytoestrogens may modulate the SBP activity and so influence the role of this protein in the delivery of hormonal information to sex steroid-dependent cells.

Topics: 4-Butyrolactone; Electrophoresis, Polyacrylamide Gel; Estradiol; Estrogens, Non-Steroidal; Genistein; Humans; Immunoelectrophoresis, Two-Dimensional; Isoflavones; Kinetics; Lignans; Masoprocol; Phytoestrogens; Plant Preparations; Plants; Sex Hormone-Binding Globulin; Structure-Activity Relationship; Testosterone

The mammalian lignans enterodiol (ED) and enterolactone (EL) produced from colonic bacterial action on dietary precursors have exhibited anticarcinogenic effects in vitro. The major lignan precursor in flaxseed (a rich source) has been identified as secoisolariciresinol diglycoside (SDG). The purpose of this study was to first isolate SDG and determine whether 1) SDG accounted for all the lignan production from flaxseed; 2) this production was dose-related; and 3) a relationship between in vitro production and in vivo urinary excretion existed. Extraction of flaxseed with dioxane:ethanol (1:1, v/v) followed by chromatographic separations yielded the purified SDG. Rats were fed a high fat diet without/with 2.5, 5 or 10 g/100g ground flaxseed or 1.1, 2.2 or 4.4 micromol SDG/d (equivalent to levels in the respective flaxseed diets) for 4 wk. In vitro lignan production was assessed by fermenting flaxseed or SDG for 24 h with human fecal inoculum. Urinary lignan excretion increased linearly with doses from 0-5% flaxseed and 0-2.2 micromol SDG/d followed by a plateau, indicating a threshold response. When all doses were considered, a curvilinear relationship was observed. A similar trend was seen in vitro for SDG, resulting in a high correlation between in vitro production and in vivo excretion of lignans (r = 0.990, P < 0.02). Thus in vivo response can be predicted with confidence based on in vitro results. Theoretical urinary ED + EL from the SDG present in flaxseed correlated with the actual excretion in flaxseed-fed animals (r = 0.655, P < 0.005). However, urinary ED + EL of SDG-fed rats was only 20% of levels of flaxseed-fed rats, indicating the presence of other precursors or incomplete conversion of SDG to ED and EL.

Topics: 4-Butyrolactone; Animals; Butylene Glycols; Colon; Dietary Fiber; Female; Glycosides; In Vitro Techniques; Lignans; Lignin; Rats; Rats, Sprague-Dawley; Regression Analysis; Seeds

Plasma levels of the lignans enterodiol and enterolactone, and also the isoflavonic phyto-oestrogens daidzein, equol and genistein, are reported for postmenopausal Australian women consuming a traditional diet supplemented with linseed, soya flour or clover sprouts. Analysis was performed by gas chromatography-mass spectrometry, after enzymatic hydrolysis and ion-exchange chromatography. Following linseed supplementation, combined levels of enterolactone and enterodiol reached 500 ng/ml, whereas after soya flour or clover sprouts the respective concentrations of equol, daidzein and genistein reached 43, 312 and 148 ng/ml. Not all subjects were able to produce equol from daidzein. The possible relationship and role of these weak dietary oestrogens as restraining factors in the development of hormone-dependent cancers in Asian populations is discussed.

Topics: 4-Butyrolactone; Australia; Chromans; Diet; Equol; Estrogens; Female; Food, Fortified; Gas Chromatography-Mass Spectrometry; Genistein; Glycine max; Humans; Isoflavones; Lignans; Linseed Oil; Middle Aged; Monoamine Oxidase Inhibitors; Neoplasms; Plants, Edible; Postmenopause

A method is presented to quantify selected mammalian lignans in human physiological fluids by gas chromatography (GC) coupled with ion mobility spectrometry (IMS). The use of IMS following GC permitted the selective and sensitive measurement of 2,3-bis(3-hydroxybenzyl)butane-1,4-diol (i.e., enterodiol) and trans-2,3-bis(3-hydroxybenzyl)-gamma-butyrolactone (i.e., enterolactone) concentrations in urine and plasma following dietary supplementation with whole wheat/flaxseed bread high in mammalian lignan precursors. Following six weeks of flaxseed feeding, urinary and plasma levels of enterodiol and enterolactone were elevated, exceeding the amounts found at baseline by a factor of 3-5. The approach to mammalian lignan methodology presented herein provides novel analytical phytochemical procedures for assessing the impact of lignan consumption in human health and disease.

Topics: 4-Butyrolactone; Adult; Animals; Body Fluids; Chromatography, Gas; Diet; Humans; Ions; Lignans; Male; Mammals; Spectrum Analysis

Mammalian lignans such as enterolactone and enterodiol, which are produced in the colon from precursors in foods, have been suggested as playing a role in the cancer-protective effect of vegetarian diets. Despite this, very little is known regarding the amount that is produced from different food products. Therefore, the objective of this study was to determine the production of mammalian lignans from 68 common plant foods by using the technique of in vitro fermentation with human fecal microbiota, which simulates colonic fermentation. Results showed a wide range (21-67,541 microgram(s)/100 g sample) in the amount of lignans produced. On the average as a group, the oilseeds produced the highest amounts (20,461 +/- 12,685), followed by the dried seaweeds (900 +/- 247), whole legumes (562 +/- 211), cereal brans (486 +/- 90), legume hulls (371 +/- 52), whole grain cereals (359 +/- 81), vegetables (144 +/- 23), and fruits (84 +/- 22). The vegetables produced the second highest concentration of lignans (1,546 +/- 280) when the data were expressed on a moisture-free basis. Flaxseed flour and its defatted meal were the highest producers of lignans (mean 60,110 +/- 7,431). Lignan production with the in vitro method related well to the urinary lignan excretion observed in rats and humans. The data should be useful in the estimation of lignan production from a given diet and in the formulation of high-lignan-producing diet for the purpose of reducing the cancer risk.

Topics: 4-Butyrolactone; Animals; Bacteria; Butylene Glycols; Chromatography, Gas; Edible Grain; Fabaceae; Feces; Fermentation; Food; Fruit; Humans; Lignans; Lignin; Mammals; Plants, Edible; Plants, Medicinal; Seaweed; Vegetables

We describe an isotope dilution gas chromatographic-mass spectrometric method for the quantitative determination of the lignans enterolactone, enterodiol and matairesinol and the isoflavonoids daidzein, equol, O-desmethylangolensin and genistein in urine. Furthermore we present the gas chromatographic/mass spectrometer identification of genistein. Urine samples were extracted on Sep-Pak cartridges, conjugated fractions were isolated by chromatography on the acetate form of DEAE-Sephadex and deuterated internal standards of all seven compounds were added to the samples before hydrolysis. The hydrolysate was extracted on a Sep-Pak cartridge and following chromatography on the acetate form of QAE-Sephadex two fractions were obtained: Fraction 1 contained equol, enterolactone, enterodiol, matairesinol and all estrogens and fraction 2 contained O-desmethylangolensin, daidzein and genistein. The latter was ready for gas chromatography/mass spectrometry, but the first one was further purified to eliminate the estrogens by chromatography on the carbonate form of QAE-Sephadex. Following silylation, the samples were analyzed by combined capillary column gas chromatography/mass spectrometry in the selective ion monitoring mode. The within-assay imprecision varied from 0.8-15.2% (mean 8.7%) and the between-assay imprecision from 4.1-13.9% (mean 9.3%), depending on compound and concentration level. The mean recovery of authentic standards added to urine extracts before hydrolysis varied from 96.6 to 105.5%. Values obtained from 10 Finnish omnivorous men are presented. Individual values for matairesinol (excretion range 3.3-59.9 nmol/24 h) and genistein (range 21.8-1180 nmol/24 h) in human urine have never been published before.

Topics: 4-Butyrolactone; Butylene Glycols; Chromans; Equol; Female; Furans; Gas Chromatography-Mass Spectrometry; Genistein; Humans; Indicator Dilution Techniques; Isoflavones; Lignans; Lignin; Magnetic Resonance Spectroscopy; Male

The binding of the lignans, enterolactone, enterodiol, nordihydroguaiaretic acid (NDGA), and the isoflavonic phytoestrogen equol, to human and rat alpha-fetoprotein (AFP) was studied. They had differential inhibitory effects (NDGA greater than equol greater than enterolactone greater than enterodiol) on the binding of estrone and estradiol to rat AFP and the binding of unsaturated fatty acid to both rat and human AFP. Inhibition was dose-dependent. The apparent dissociation constants (Kd) for phytoestrogens binding to AFP were: Kd NDGA = 5 +/- 1.2.10(-7) M, Kd equol = 6.7 +/- 0.8.10(-6) M, Kd enterolactone = 1.7 +/- 0.4.10(-5) M and Kd enterodiol = 2.2 +/- 0.6.10(-5) M. The Kd for estrone binding to rat AFP was increased by increasing concentrations of equol, but the number of esterone binding sites remained unchanged. This, plus the results of double-reciprocal plots, suggests that they compete for the same site(s). NDGA also competitively inhibited estrone binding at low NDGA concentrations (increased Kd), but high concentrations induced conformational changes in rat AFP, as both Kd and the number of binding sites (n) were altered. Both rat and human AFPs underwent changes in electrophoretic behaviour and loss of immunoreactivity with increasing NDGA, suggesting that NDGA binding induces conformational changes in the AFPs. However, equol did not alter the electrophoretic or immunological properties of either rat or human AFP, providing further evidence for qualitative differences in the effects of these diphenols. These findings indicate that phytoestrogens could play a role in AFP-dependent normal and pathological growth and development.

Topics: 4-Butyrolactone; Adult; alpha-Fetoproteins; Animals; Arachidonic Acid; Binding, Competitive; Butylene Glycols; Chromans; Equol; Estrogens; Estrogens, Non-Steroidal; Humans; Immunoelectrophoresis; Isoflavones; Ligands; Lignans; Masoprocol; Phytoestrogens; Plant Preparations; Rats; Rats, Inbred Strains

The effect of each of twelve mammalian lignan derivatives on the growth of human mammary tumor ZR-75-1 cells was examined. At a concentration less than 10 micrograms/ml, tumor cell growth was inhibited from 18-68%. The effect of 2,3-dibenzylbutane-1,4-diol(hattalin) was found to be strongest, inhibiting growth by 50% at a concentration (EC50) of 2.1 micrograms/ml. Hattalin inhibited membrane Na+, K(+)-ATPase of canine kidney cortex. It also inhibited the ATPase of the plasma membrane fraction from both cultured cells and a section of human breast cancer tissue at a concentration ranging from 0.5 to 2.0 mM. However, only a few percent of membrane ATPase from either ZR-75-1 cells or breast carcinoma tissue was inhibited by 2.0 mM of ouabain, suggesting that the target ATPase of hattalin was other than ouabain-sensitive ATPase. The relative incorporation of [3H]thymidine per 1 x 10(5) cells into the acid-precipitable fraction of ZR-75-1 cells was not affected by 1-50 micrograms/ml of hattalin, while a marked decrease resulted from 1-10 micrograms/ml of 5-fluorouracil (5-FU). These results suggest that the suppressive effect of hattalin on tumor cell growth may not occur through inhibition of DNA synthesis but rather partly by inhibition of the plasma membrane ATPase other than Na+ and K(+)-dependent ones.

Topics: 4-Butyrolactone; Adenosine Triphosphatases; Animals; Benzyl Compounds; Breast Neoplasms; Butylene Glycols; Cell Division; Cell Line; Dogs; Dose-Response Relationship, Drug; Fluorouracil; Humans; Kidney Cortex; Lethal Dose 50; Lignans; Ouabain

The phenolic lignans enterolactone and enterodiol appear periodically in women's urine, dependent upon synthesis from plant-derived lignans by the intestinal microflora. The phytoestrogen equol is also present in women's urine, and is also derived from a vegetarian diet. Antiestrogenic or antiproliferative actions of these compounds have been postulated and related to the observation that there is a reduced incidence of breast cancer associated with diet. We evaluated the estrogenic and antiestrogenic activity of these compounds using four sensitive assays in tissue culture, including the use of human breast cancer cell lines T47D and MCF-7. Unexpectedly, we found that enterolactone and enterodiol, as well as equol, are weak estrogens, and that enterolactone and equol could stimulate the growth of estrogen-dependent breast cancer cell lines. We suggest that these environmental agents can promote the growth of breast cancer, particularly hormone-dependent metastases that may be located near the gut or in the mesenteries or liver, where the concentration of these intestinally produced compounds would be highest. Treatment with an antiestrogen such as tamoxifen blocks the estrogenic activity of these compounds. In the absence of treatment with an antiestrogen such as tamoxifen, hormonal therapy to block steroidal estrogen synthesis in a patient with breast cancer could conceivably be circumvented by a vegeterian diet rich in the precursors to estrogenic compounds such as enterolactone and equol.

Topics: 4-Butyrolactone; Benzopyrans; Breast Neoplasms; Butylene Glycols; Cell Division; Chromans; Equol; Estrogens; Female; Furans; Humans; Isoflavones; Lignans; Receptors, Progesterone; Tumor Cells, Cultured

It was recently observed that the urinary excretion of animal lignans is low in postmenopausal breast cancer patients compared to normal omnivorous and vegetarian women. In addition, the mean excretion of the isoflavonic phytoestrogen equol tended to be lower. Because nonhuman primates appear to be remarkably resistant to the carcinogenic effect of estrogens, we investigated the possible occurrence of lignans and phytoestrogens in the urine of chimpanzees on their regular diet. Five major diphenols were isolated and identified by capillary gas chromatography and mass spectrometry by comparison with synthesized authentic reference compounds. Three of these compounds, the phytoestrogen equol and its precursor daidzein, the lignan enterolactone, were according to preliminary assays excreted in very large amounts. In addition, the lignan enterodiol and the daidzein metabolite O-desmethylangolensin were identified. It is concluded that the chimpanzee excretes both isoflavonic phytoestrogens and lignans in urine, apparently in high concentrations. It is suggested that these compounds may play a role in the maintenance of the resistance against carcinogenic effects of estrogens, which nonhuman primates possess, because both equol and enterolactone have been shown to have antiestrogenic properties in animals. However, much further work is necessary before the possible biological role of these compounds may be established.

Topics: 4-Butyrolactone; Animals; Butylene Glycols; Chromans; Diet; Equol; Estrogens; Estrogens, Non-Steroidal; Isoflavones; Lignans; Male; Pan troglodytes; Phytoestrogens; Plant Extracts; Plant Preparations

Lignans have, until recently, been found only in plants. Enterolactone and enterodiol are the major lignans present in the urine of humans and have a potential physiological protective role against cancer. It has been shown that these compounds can be formed in vitro by human faecal flora and that enterodiol is oxidized to enterolactone by bacteria that are present in stools at a concentration of up to 10(3)/g. It was also possible to produce both of these lignans in vitro from linseeds and from secoisolariciresinol, a precursor present in linseed, by bacteria present in stools, at a concentration of between 10(3) and 10(4)/g. Enterolactone was produced from matairesinol, a more abundant plant lignan than secoisolariciresinol, after incubation with a mixed faecal flora under both aerobic and anaerobic conditions. In each case conversion was dependent on the presence of viable bacteria. These findings indicate that a number of different pathways operate to produce enterolactone and enterodiol depending on the ingested dietary precursor.

Topics: 4-Butyrolactone; Bacteria; Butylene Glycols; Clostridium; Feces; Humans; Lignans; Linseed Oil; Metronidazole; Plant Extracts

Methods are described to extract and measure 2,3-bis(3-hydroxybenzyl)-gamma-butyrolactone (enterolactone) and 2,3-bis(3-hydroxybenzyl)butane-1,4-diol (enterodiol) from physiological fluids. They are based on the use of stable labelled analogues of each compound as internal standards with end point assay being by gas chromatography mass spectrometry. The methods were developed to quantify enterolactone and enterodiol in experiments designed to investigate the significance and source of these compounds in man and animals. Examples of studies on human ovarian blood and investigations of their excretion in urine following dietary manipulation are described.

Topics: 4-Butyrolactone; Adult; Animals; Butylene Glycols; Diet; Female; Furans; Gas Chromatography-Mass Spectrometry; Haplorhini; Humans; Hydrolysis; Lignans; Middle Aged

Dietary studies and assays of urinary lignans in postmenopausal women showed that lignan excretion is significantly lower in urine of women with breast cancer than in normal omnivorous and vegetarian women and confirmed that there is a significant correlation between fibre intake and lignan excretion. It is suggested that the precursors of the human lignans enterolactone and enterodiol formed by the intestinal microflora are to be found in fibre-rich foods such as grains, nuts, and legumes. Excretion of equol, which has antioestrogenic properties, was similar in all groups studied and did not correlate with fibre intake, but occasional high values were found in some subjects.

Topics: 4-Butyrolactone; Aged; Benzopyrans; Breast Neoplasms; Butylene Glycols; Chromans; Diet; Diet, Vegetarian; Dietary Fiber; Energy Intake; Equol; Female; Furans; Humans; Isoflavones; Lignans; Menopause; Menstruation; Middle Aged; Plant Extracts

Topics: 4-Butyrolactone; Animals; Biotransformation; Butylene Glycols; Diet; Enterobacteriaceae; Furans; Lignans; Plant Extracts; Rats; Seeds

We describe a capillary column gas chromatographic (GC) method for the analysis of lignans in urine. Lignans are excreted as mono-glucuronides which are first extracted on a small reversed-phase cartridge of octadecylsilane bonded silica (Sep-Pak C18) and thereafter isolated by anion exchange chromatography on DEAE-Sephadex A-25, prepared in the acetate form. Lignan mono-glucuronides are enzymatically hydrolysed and re-extracted on a Sep-Pak C18 cartridge. Quantification is carried out by capillary column GC of the trimethylsilyl ether derivatives. The specificity of the method was checked by GC/MS and the intra-assay coefficient of variation (CV) for the two lignans, enterolactone (trans-2,3-bis(3-hydroxybenzyl)butyrolactone) and enterodiol (2.3-bis(3-hydroxybenzyl)butane-1,4-diol) varied between 5 and 8%. Some values for the excretion of these lignans by normal men and women are presented.

Topics: 4-Butyrolactone; Adult; Butylene Glycols; Chromatography, Gas; Chromatography, Ion Exchange; Female; Gas Chromatography-Mass Spectrometry; Glucuronates; Humans; Lignans; Male; Middle Aged; Plant Extracts

Topics: 4-Butyrolactone; Bacteria; Butylene Glycols; Clostridium; Depression, Chemical; Female; Humans; Intestines; Lignans; Male; Menstruation; Metronidazole; Neoplasms; Oxytetracycline; Plant Extracts; Pregnancy

Topics: 4-Butyrolactone; Animals; Bile; Butylene Glycols; Enterobacteriaceae; Enterohepatic Circulation; Female; Gas Chromatography-Mass Spectrometry; Germ-Free Life; Humans; Lignans; Male; Plant Extracts; Rats

The definitive identification of the first lignans to be found in humans and animals is described. Gas chromatography--mass spectrometry, n.m.r. spectroscopy, i.r. spectroscopy and chemical techniques were employed to establish the structures of two lignans as trans-2,3-bis(3-hydroxybenzyl)-gamma-butyrolactone and 2,3-bis(3-hydroxybenzyl)butane-1,4-diol. Both compounds are essetially racemic. Evidence was also found for several methoxy analogues of these lignans in the vervet monkey.

Topics: 4-Butyrolactone; Animals; Butylene Glycols; Chemical Phenomena; Chemistry; Chlorocebus aethiops; Chromatography, Gas; Chromatography, Thin Layer; Female; Furans; Gas Chromatography-Mass Spectrometry; Humans; Lignans; Magnetic Resonance Spectroscopy; Mass Spectrometry; Pregnancy

Topics: 4-Butyrolactone; Adult; Age Factors; Butylene Glycols; Chromatography, Gas; Female; Furans; Glucuronates; Humans; Lignans; Male; Mass Spectrometry; Middle Aged; Plant Extracts; Pregnancy; Sex Factors