lignans and secoisolariciresinol-diglucoside

Hyperlipidemia, high levels of blood lipids including cholesterol and triglycerides, is a major risk factor for cardiovascular disease. Traditional treatments of hyperlipidemia often include lifestyle changes and pharmacotherapy. Recently, flaxseed has been approved as a nutrient that lowers blood lipids. Several metabolites of flaxseed lignan secoisolariciresinol diglucoside (SDG), have been identified that reduce blood lipids. SDG is present in flaxseed hull as an ester-linked copolymer with 3-hydroxy-3-methylglutaric acid (HMGA). However, purification processes involved in hydrolysis of the copolymer and enriching SDG are often expensive. The natural copolymer of SDG with HMGA (SDG polymer) is a source of bioactive compounds useful in prophylaxis of hypercholesterolemia. After consumption of the lignan copolymer, SDG and HMGA are released in the stomach and small intestines. SDG is metabolized to secoisolariciresinol, enterolactone and enterodiol, the bioactive forms of mammalian lignans. These metabolites are then distributed throughout the body where they accumulate in the liver, kidney, skin, other tissues, and organs. Successively, these metabolites reduce blood lipids including cholesterol, triglycerides, low density lipoprotein cholesterol, and lipid peroxidation products. In this review, the metabolism and efficacies of flaxseed-derived enriched SDG and SDG polymer will be discussed.

Topics: Animals; Cholesterol; Flax; HMGA Proteins; Humans; Hyperlipidemias; Lignans; Lipids; Mammals; Polymers; Triglycerides

Flaxseed is a rich source of the omega-3 fatty acid, alpha linolenic acid, the lignan secoisolariciresinol diglucoside and fiber. These compounds provide bioactivity of value to the health of animals and humans through their anti-inflammatory action, anti-oxidative capacity and lipid modulating properties. The characteristics of ingesting flaxseed or its bioactive components are discussed in this article. The benefits of administering flaxseed or the individual bioactive components on health and disease are also discussed in this review. Specifically, the current evidence on the benefits or limitations of dietary flaxseed in a variety of cardiovascular diseases, cancer, gastro-intestinal health and brain development and function, as well as hormonal status in menopausal women, are comprehensive topics for discussion.

Topics: alpha-Linolenic Acid; Animals; Anti-Inflammatory Agents; Antioxidants; Brain; Butylene Glycols; Cardiovascular Diseases; Diet; Dietary Fiber; Dietary Supplements; Fatty Acids, Omega-3; Female; Flax; Functional Food; Gastrointestinal Diseases; Glucosides; Hormones; Humans; Lignans; Lipid Metabolism; Neoplasms; Seeds

Lignans are a group of phytonutrients which are widely distributed in the plant kingdom. Flaxseed is the richest source of providing lignan precursor such as secoisolariciresinol diglucoside (SDG). This article reviews the studies relevant to experimental models in animals and humans demonstrating the possible nutraceutical actions of SDG to prevent and alleviate lifestyle-related diseases. A local and international web-based literature review for this project was carried out to provide information relating to the study. The major key word "SDG" was selected to gather information using the electronic databases pertaining to the current state of flaxseed lignans composition, bioactive compounds, metabolism and to find out their role in terms of chemopreventive action. The extraction methods vary from simple to complex depending on separation, fractionation, identification and detection of the analytes. The majority of studies demonstrate that SDG interferes with the development of different types of diseases like cardiovascular, diabetic, lupus nephritis, bone, kidney, menopause, reproduction, mental stress, immunity, atherosclerosis, hemopoietic, liver necrosis and urinary disorders due to its various biological properties including anti-inflammatory, antioxidant, antimutagenic, antimicrobial, antiobesity, antihypolipidemic and neuroprotective effects. Moreover, SDG has a defending mediator against various cancers by modulating multiple cell signaling pathways. As discussed in this review, SDG has shown therapeutic potential against a number of human diseases and can be recommended for discerning consumers.

Topics: Animals; Antioxidants; Butylene Glycols; Chemoprevention; Disease Models, Animal; Flax; Glucosides; Humans; Lignans; Phytochemicals; Plant Extracts

Many natural products, including vitamin E, garlic, purpurogallin, flaxseed and its components [secoisolariciresinol diglucoside (SDG) and flax lignan complex (FLC)] and resveratrol have been reported to suppress hypercholesterolemic atherosclerosis. It is known that all of the drugs that suppress the development of atherosclerosis do not regress and/or slow the progression of atherosclerosis. To be of potential benefit in patients with established atherosclerosis, a drug should produce regression and/or slow the progression of atherosclerosis. In this review, the effects of vitamin E, SDG and FLC in the regression and slowing of progression of hypercholesterolemic atherosclerosis and their mechanisms have been described. The effectiveness of vitamin E in patients with established coronary disease is very controversial. However, in experimental animal controlled studies, vitamin E does not regress or slow the progression of hypercholesterolemic atherosclerosis. The mechanisms of the ineffectiveness of vitamin E in regression and slowing of progression of atherosclerosis have been discussed. SDG is effective in slowing the progression of atherosclerosis and partially effective in regression of hypercholesterolemic atherosclerosis. These effects are associated with reduction in oxidative stress. FLC does not regress hypercholesterolemic atherosclerosis but slows the progression of hypercholesterolemic atherosclerosis. Slowing of progression is associated with reduction on oxidative stress. In conclusion, vitamin E does not regress or slow the progression of established atherosclerosis. SDG slows the progression and regresses established atherosclerosis. FLC does not regress but slows the progression of established atherosclerosis.

Topics: Animals; Antioxidants; Atherosclerosis; Biological Products; Butylene Glycols; Disease Progression; Flax; Glucosides; Humans; Hypercholesterolemia; Lignans; Oxidative Stress; Phytotherapy; Vitamin E

Plant lignans are phenolic compounds generally containing a dibenzylbutane skeleton. Secoisolariciresinol diglucoside (SDG) is the major lignan found in flaxseed. SDG is known to have antioxidant and anticancer properties. SDG can potentially be used as a natural antioxidant in foods thereby preventing further oxidation reactions and thus enhance the shelf life of foods. This article reviews the patents that are concerned with the extraction of SDG from flaxseed, the richest plant source of lignans. Most of the patented techniques for the extraction, isolation, and purification of SDG are conducted on defatted flaxseed and whole flaxseed. Flaxseed hull is potentially a good starting material. Furthermore, most methods use aliphatic alcohols (methanol, ethanol, isopropanol, butanol) to extract the complexed form of SDG. Combinations of these solvents are commonly used with water. Alkaline hydrolysis liberates SDG from its complexed form. SDG is enriched by a process involving either liquid-liquid partitioning or passing the aqueous phase through anion exchange resins or C18 resins. The SDG is recovered after evaporation of the water. Analytical HPLC coupled with mass spectrometry is performed to determine the quantity and purity of the extracted SDG.

Topics: Antioxidants; Butylene Glycols; Chromatography, High Pressure Liquid; Flax; Glucosides; Humans; Lignans; Mass Spectrometry; Patents as Topic

Topics: Adult; Breast Neoplasms; Butylene Glycols; Female; Flax; Follow-Up Studies; Glucosides; Humans; Hyperplasia; Lignans; Middle Aged; Pilot Projects; Premenopause; Prognosis; Risk Factors; Young Adult

A flaxseed lignan extract containing 33% secoisolariciresinol diglucoside (SDG) was evaluated for its ability to alleviate lower urinary tract symptoms (LUTS) in 87 subjects with benign prostatic hyperplasia (BPH). A randomized, double-blind, placebo-controlled clinical trial with repeated measurements was conducted over a 4-month period using treatment dosages of 0 (placebo), 300, or 600 mg/day SDG. After 4 months of treatment, 78 of the 87 subjects completed the study. For the 0, 300, and 600 mg/day SDG groups, respectively, the International Prostate Symptom Score (IPSS) decreased -3.67 +/- 1.56, -7.33 +/- 1.18, and -6.88 +/- 1.43 (mean +/- SE, P = .100, < .001, and < .001 compared to baseline), the Quality of Life score (QOL score) improved by -0.71 +/- 0.23, -1.48 +/- 0.24, and -1.75 +/- 0.25 (mean +/- SE, P = .163 and .012 compared to placebo and P = .103, < .001, and < .001 compared to baseline), and the number of subjects whose LUTS grade changed from "moderate/severe" to "mild" increased by three, six, and 10 (P = .188, .032, and .012 compared to baseline). Maximum urinary flows insignificantly increased 0.43 +/- 1.57, 1.86 +/- 1.08, and 2.7 +/- 1.93 mL/second (mean +/- SE, no statistical significance reached), and postvoiding urine volume decreased insignificantly by -29.4 +/- 20.46, -19.2 +/- 16.91, and -55.62 +/- 36.45 mL (mean +/- SE, no statistical significance reached). Plasma concentrations of secoisolariciresinol (SECO), enterodiol (ED), and enterolactone (EL) were significantly raised after the supplementation. The observed decreases in IPSS and QOL score were correlated with the concentrations of plasma total lignans, SECO, ED, and EL. In conclusion, dietary flaxseed lignan extract appreciably improves LUTS in BPH subjects, and the therapeutic efficacy appeared comparable to that of commonly used intervention agents of alpha1A-adrenoceptor blockers and 5alpha-reductase inhibitors.

Topics: Aged; Aged, 80 and over; Butylene Glycols; Diet; Double-Blind Method; Flax; Glucosides; Humans; Lignans; Male; Middle Aged; Phytotherapy; Placebos; Plant Extracts; Prostatic Hyperplasia; Quality of Life; Urinary Tract; Urination; Urine

A lignan complex rich in the plant lignan secoisolariciresinol diglucoside (SDG) was isolated from flaxseed. SDG is metabolized by the colonic microflora to the mammalian lignans enterodiol (END) and enterolactone (ENL), and was hypothesized to reduce plasma lipid concentrations and improve antioxidant capacity. The aim of this study was to investigate the effects of a lignan complex, providing 500 mg/d of SDG, on serum concentration and urinary excretion of ENL, plasma lipids, serum lipoprotein oxidation resistance, and markers of antioxidant capacity. Healthy postmenopausal women (n = 22) completed a randomized, double-blind, placebo-controlled, crossover study. Women consumed daily a low-fat muffin, with or without a lignan complex, for 6 wk, separated by a 6-wk washout period. Serum ENL concentration, urinary ENL excretion, plasma concentrations of total cholesterol (TC), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), triacylglycerol (TAG), serum lipoprotein oxidation lag time, plasma Trolox-equivalent antioxidant capacity (TEAC), and ferric reducing ability of plasma (FRAP) were measured at the beginning and end of each intervention period. ENL concentrations in serum (P < 0.001) and ENL urinary excretion (P < 0.001) were significantly higher after the lignan complex intervention period compared with placebo. Plasma concentrations of TC, LDL-C, HDL-C, TAG, lipoprotein oxidation lag time, TEAC and FRAP were not affected. Daily consumption for 6 wk of a low-fat muffin enriched with a lignan complex significantly increased serum ENL concentrations and urinary ENL excretion in healthy postmenopausal women, but had no effect on plasma lipid concentrations, serum lipoprotein oxidation resistance, or plasma antioxidant capacity.

Topics: 4-Butyrolactone; Butylene Glycols; Cross-Over Studies; Denmark; Double-Blind Method; Female; Flax; Glucosides; Humans; Lignans; Lipids; Middle Aged; Postmenopause

Linseeds are a rich source of lignans, secondary plant substances which are suggested to possess chemopreventive effects inter alia with regard to breast cancer. In a randomised controlled trial 40 German women were informed about "5-a-day" and encouraged to increase their dietary intake of fruit and vegetables. Moreover 19 participants were randomly assigned to an intervention group supplemented with ground linseeds (20 g/d) over a 2-month period. Before and after intervention, urine and blood samples were collected after an overnight fast. Analysis was by intention-to-treat and the outcome parameters of interest were enterolignan concentrations. After linseed supplementation, enterolignan concentrations (mean) measured as their glucuronides by a newly developed high performance liquid chromatography electrospray mass spectrometry (HPLC-ESI-MS) in serum (122 nmol/l) as well as in urine (72 micromol/l) showed a significant increase (P<0.01) compared to pre-intervention values (47 nmol/l and 29 micromol/l). In the control group enterolignan levels were raised slightly but did not reach significance. Serum and urinary enterolignans of the whole collective showed a good pairwise correlation.

Topics: Adult; Butylene Glycols; Chromatography, High Pressure Liquid; Diet; Dietary Supplements; Female; Fruit; Germany; Glucosides; Humans; Lignans; Linseed Oil; Middle Aged; Spectrometry, Mass, Electrospray Ionization; Vegetables

Atopic dermatitis (AD) is a severe inflammatory skin disease caused by a combination of genetic, immune, and environmental factors. Intestinal microbiome disorders and changes in the immune microenvironment are associated with AD. We observed that gut bacterial metabolite enterolactone (ENL) was significantly reduced in AD model mice. Notably, patients with early childhood-onset AD exhibited decreased sera ENL level compared to the healthy controls, and the ENL level was negatively correlated with the SCORAD index. Secoisolariciresinol-diglycoside (SDG) is a natural dietary lignan of flaxseeds that can be converted by intestinal bacteria to ENL. Repeated applications of 2,4-dinitrochlorobenzene (DNCB) were performed on the ear and dorsal skin of mice to induce AD-like symptoms and skin lesions. Oral administration of SDG significantly decreased serum IgE levels and limited skin inflammation in the DNCB-induced AD mice. In addition, SDG treatment strongly limited the Th2 responses in AD mice. Moreover, we demonstrated that the IL-4 production was significantly suppressed by ENL under Th2 polarization conditions via the JAK-STAT6 signaling pathway in a concentration-dependent manner. We concluded that SDG and its derived metabolite ENL ameliorated AD development by reducing the Th2 immune response. These results suggested that SDG and ENL might be exploited as potential therapeutic candidates for AD treatment.

Topics: 4-Butyrolactone; Animals; Anti-Inflammatory Agents; Butylene Glycols; Child, Preschool; Dermatitis, Atopic; Dinitrochlorobenzene; Glucosides; Humans; Immunity; Lignans; Mice; Mice, Inbred BALB C; Skin

Cytokine storms lead to cardiovascular diseases (CVDs). Natural herbal compounds are considered the primary source of active agents with the potential to prevent or treat inflammatory-related pathologies such as CVD and diabetes. Flaxseed contains phytochemicals, including secoisolariciresinol diglucoside (SDG), α-linolenic acid (ALA), and lignans, termed "SAL." Hence, we evaluated the effect of the SAL on the H9c2 cardiac cells in hyperlipidemic and hyperglycemic conditions. Here, candidate hub genes, TNF-α, IL6, SIRT1, NRF1, NPPA, and FGF7, were selected as effective genes in diabetic cardiovascular pathogenesis based on in-silico analysis and chemoinformatic. Myocardial infarction (MI) was induced using H9c2 cardiac cells in hyperlipidemic and hyperglycemic conditions. Real-time qPCR was conducted to assess the expression level of hub genes. This study indicated that SAL compounds bound to the Il-6, SIRT1, and TNF-α active sites as druggable candidate proteins based on the chemoinformatics analysis. This study displayed that the TNF-α, IL6, SIRT1, NRF1, NPPA, and FGF7 network dysfunction in MI models were ameliorated by SAL consumption. Furthermore, SAL compounds improved the function and myogenesis of H9c2 cells in hyperlipidemic and hyperglycemic conditions. Our data suggested that phytochemicals obtained from flaxseed might have proposed potential complementary treatment or preventive strategies for MI. PRACTICAL APPLICATIONS: Phytochemicals obtained from flaxseed (SAL) could reverse diabetic heart dysfunction hallmarks and provide new potential treatment approaches in cardiovascular therapy. SAL could be considered complementary and alternative medicines for treating various disorders/diseases singly or synchronizing with prescription drugs.

Topics: alpha-Linolenic Acid; Computational Chemistry; Diabetes Mellitus; Flax; Interleukin-6; Lignans; Pharmacophore; Phytochemicals; Sirtuin 1; Tumor Necrosis Factor-alpha

Secoisolariciresinol diglucoside (SDG) is isolated from

Topics: Breast Neoplasms; Butylene Glycols; Female; Flax; Glucosides; Humans; Lignans; MCF-7 Cells

LGM2605 is a synthetic version of the naturally occurring flaxseed lignan secoisolariciresinol diglucoside (SDG), with known anti-inflammatory and antioxidant properties; however, its effects on gut microbial composition have not previously been evaluated. In the present study, we sought to determine how the 10-day oral administration of LGM2605 alters the gut microbiota of mice. Eight-week-old female C57BL/6 mice were treated with either LGM2605 or saline, administered daily via oral gavage over a 10-day treatment period. Upon termination of treatment, mouse cecums (n = 31) were collected, and cecal DNA was isolated. 16S rRNA genes were sequenced and analyzed in Mothur to identify changes in gut microbial composition induced by LGM2605 treatment (v. saline control). We then assessed community composition, performed indicator taxa analysis, and measured alpha and beta diversity. Overall, LGM2605 significantly altered the gut microbiota of mice; we reported alterations in 3 bacterial phyla and 22 genera as a result of treatment. The study here identifies for the first time significant alterations in the gut microbiota of mice following oral administration of LGM2605, in general shifting toward a more anti-inflammatory composition. These findings lay the foundation for future investigations utilizing LGM2605 to control gut dysbiosis and, by extension, systemic inflammation.

Topics: Administration, Oral; Animals; Anti-Infective Agents; Biodiversity; Butylene Glycols; Cecum; Computational Biology; Dysbiosis; Female; Flax; Gastrointestinal Microbiome; Glucosides; Lignans; Mice; Mice, Inbred C57BL; RNA, Ribosomal, 16S; Seeds

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

Visual function depends on the intimate structural, functional and metabolic interactions between the retinal pigment epithelium (RPE) and the neural retina. The daily phagocytosis of the photoreceptor outer segment tips by the overlaying RPE provides essential nutrients for the RPE itself and photoreceptors through intricate metabolic synergy. Age-related retinal changes are often characterized by metabolic dysregulation contributing to increased lipid accumulation and peroxidation as well as the release of proinflammatory cytokines. LGM2605 is a synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant and anti-inflammatory properties demonstrated in diverse in vitro and in vivo inflammatory disease models. In these studies, we tested the hypothesis that LGM2605 may be an attractive small-scale therapeutic that protects RPE against inflammation and restores its metabolic capacity under lipid overload. Using an in vitro model in which loss of the autophagy protein, LC3B, results in defective phagosome degradation and metabolic dysregulation, we show that lipid overload results in increased gasdermin cleavage, IL-1 β release, lipid accumulation and decreased oxidative capacity. The addition of LGM2605 resulted in enhanced mitochondrial capacity, decreased lipid accumulation and amelioration of IL-1 β release in a model of defective lipid homeostasis. Collectively, these studies suggest that lipid overload decreases mitochondrial function and increases the inflammatory response, with LGM2605 acting as a protective agent.

Topics: Antioxidants; Autophagy; Butylene Glycols; Cell Line; Cytokines; Gene Expression; Glucosides; Humans; Inflammation; Lignans; Lipid Metabolism; Lipids; Mitochondria; Oxidation-Reduction; Oxidative Stress; Phagocytosis; Phagosomes; Retinal Pigment Epithelium; Retinal Pigments

Gut microbial processing of dietary flaxseed (FS) contributes to its health benefits, but the relative effects of its bioactive components (lignans, omega-3 fatty acids, fiber) on the microbiota are unclear. We investigated the gut microbial compositional and functional responses to whole FS and its isolated components, FS oil (FSO) and secoisolariciresinol diglucoside (SDG) (precursor to microbial-derived enterolignans) to help understand their contribution to whole FS benefits. Cecum content and fecal samples were collected from C57BL/6 female mice fed a basal diet (AIN93G) or isocaloric diets containing 10% FS or 10% FS-equivalent amounts of FSO or SDG for 21 days. Cecal and fecal microbiota composition and predicted genomic functions, and their relationship with serum enterolignans were evaluated. Only FS modified the community structure. Shared- and diet-specific enriched taxa and functions were identified. Carbohydrate and protein processing functions were enriched in FS mice, and there was a positive correlation between select enriched taxa, encompassing fiber degraders and SDG metabolizers, and serum enterolignans. This was not observed in mice receiving isolated FSO and SDG, suggesting that FS fiber supports SDG microbial metabolism. In conclusion, the cooperative activities of a diverse microbiota are necessary to process FS components and, when administered at the amount present in FS, these components may act together to affect SDG-derived enterolignans production. This has implications for the use of FS, FSO and SDG in clinical practice.

Topics: Animals; Butylene Glycols; Cecum; Diet; Dietary Fiber; Fatty Acids, Omega-3; Feces; Female; Flax; Gastrointestinal Microbiome; Glucosides; Lignans; Linseed Oil; Mice; Mice, Inbred C57BL

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

Exposure to the polyphenolic plant lignan secoisolariciresinol diglucoside (SDG) and its metabolite enterolactone (ENL) has been associated with reduced breast cancer progression, particularly for estrogen receptor alpha (ERα)-negative disease, and decreased preclinical mammary tumor growth. However, while preclinical studies have established that SDG and ENL affect measures of progression in models of triple-negative breast cancer (TNBC, a subset of ERα-negative disease), the molecular mechanisms underlying these effects remain unclear.. C57BL/6 mice were fed a control diet (control, 10% kcal from fat) or control diet + SDG (SDG, 100 mg/kg diet) for 8 weeks, then orthotopically injected with syngeneic E0771 mammary tumor cells (a model of TNBC); tumor growth was monitored for 3 weeks. The role of reduced NF-κB signaling in SDG's anti-tumor effects was explored in vitro via treatment with the bioactive SDG metabolite ENL. In addition to the murine E0771 cells, the in vitro studies utilized MDA-MB-231 and MCF-7 cells, two human cell lines which model the triple-negative and luminal A breast cancer subtypes, respectively.. SDG supplementation in the mice significantly reduced tumor volume and expression of phospho-p65 and NF-κB target genes (P < 0.05). Markers of macrophage infiltration were decreased in the distal-to-tumor mammary fat pad of mice supplemented with SDG relative to control mice (P < 0.05). In vitro, ENL treatment inhibited viability, survival, and NF-κB activity and target gene expression in E0771, MDA-MB-231, and MCF-7 cells (P < 0.05). Overexpression of Rela attenuated ENL's inhibition of E0771 cell viability and survival.. SDG reduces tumor growth in the E0771 model of TNBC, likely via a mechanism involving inhibition of NF-κB activity. SDG could serve as a practical and effective adjuvant treatment to reduce recurrence, but greater understanding of its effects is needed to inform the development of more targeted recommendations for its use.

Topics: 4-Butyrolactone; Animals; Anti-Inflammatory Agents; Biomarkers; Butylene Glycols; Cell Line, Tumor; Cell Survival; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Flax; Gene Expression Profiling; Glucosides; Immunohistochemistry; Lignans; Mammary Neoplasms, Animal; Mice; NF-kappa B; Signal Transduction

An ultra performance liquid chromatography electrospray ionization high-resolution mass spectrometry (UPLC/ESI-HRMS) method was developed and validated for simultaneous quantification of cyanogenic glycosides (CGs), [linustatin (LIS) and neolinustatin (NLIS)], and the main lignan, secoisolariciresinol diglucoside (SDG) in Linoforce® (LF) [flaxseed (Linum usitatissimum L.) coated with two herbal extracts (Senna alexandrina mill and Frangula alnus)]. CGs and SDG were extracted from defatted ground LF by a new procedure consisting of an aqueous methanol ultrasound-assisted extraction followed by an aqueous alkaline ultrasound-assisted extraction of the residue. The combined extracted solutions were then hydrolyzed by 0.02 M NaOH to release SDG from its hydroxymethyl glutaryl ester-linked complex (SDG-HMG). After hydrolysis, the sample was acidified and analyzed directly, without the need of any additional clean-up steps, by UPLC/ESI-HRMS in positive mode. The identification of CGs and SDG was confirmed by the similar retention time and similar MS spectra to the corresponding authentic standards. The quantification was performed using the corresponding extracted ion chromatograms and amygdalin as internal standard. The overall method was validated in terms of linearity, stability, selectivity, precision and accuracy. The developed method was successfully applied to the quantification of CGs and SDG in LF and also in non-coated flaxseed. This is the first report on the simultaneous quantification of CGs and SDG in LF and flaxseed.

Topics: Amygdalin; Butylene Glycols; Chemistry Techniques, Analytical; Chromatography, High Pressure Liquid; Flax; Glucosides; Glycosides; Hydrolysis; Lignans; Mass Spectrometry; Seeds

Recent studies have shown that the high susceptibility of flaxseed oil nanoemulsions to lipid oxidation limits their incorporation into functional foods and beverages. For this reason, the impact of various flaxseed phenolic extracts on the physical and oxidative stability of flaxseed oil nanoemulsions was investigated. Flaxseed lignan extract (FLE) and secoisolariciresinol (SECO) exhibited antioxidant activity whereas secoisolariciresinol diglucoside (SDG) and p-coumaric acid (CouA) exhibited prooxidant activity in the flaxseed oil nanoemulsions. The antioxidant potential of flaxseed phenolics in the nanoemulsions was as follows: SECO < CouA < SDG ≈ FLE. Moreover, the antioxidant/prooxidant activity of the phenolics was also related to their free radical scavenging activity and partitioning in the nanoemulsions. Our results suggested that both SECO and FLE were good plant-based antioxidants for improving the stability of flaxseed oil nanoemulsions.

Topics: Antioxidants; Butylene Glycols; Emulsions; Flax; Glucosides; Hydrolysis; Lignans; Linseed Oil; Nanostructures; Oxidation-Reduction; Plant Extracts; Polyphenols; Water

Hairy root culture is a promising alternative method for the production of secondary metabolites. In this study, transformed root of Linum usitatissimum was established using Agrobacterium rhizogenes A4 strain from root cultures for lignans, phenolic acids and antioxidant capacity determination. Total lignin content (secoisolariciresinol diglucoside, secoisolariciresinol and matairesinol) was 55.5% higher in transformed root cultures than in the non-transformed root culture. Secoisolariciresinol was detected in higher concentration (2.107 μmol/g DM) in the transformed root culture than non-transformed culture (1.099 μmol/g DM). Secoisolariciresinol diglucoside and matairesinol were exclusively detected in the transformed root culture, but were not found in the non-transformed root culture. The overall production of phenolic acids in transformed roots was approximately 3.5 times higher than that of the corresponding non-transformed culture. Free radical scavenging DPPH˙ and ABTS˙

Topics: Antioxidants; Biphenyl Compounds; Butylene Glycols; Flax; Furans; Glucosides; Hydroxybenzoates; Lignans; Picrates; Plant Roots; Tissue Culture Techniques

The multipurpose plant species Linum usitatissimum famous for producing linen fibre and containing valuable pharmacologically active polyphenols, has rarely been tested for it's in vitro biosynthesis potential of lignans and neolignans. The current study aims at the synergistic effects of mineral nutrients variation and different photoperiod treatments on growth kinetics and biomass accumulation in in vitro cultures of Linum usitatissimum. Both nutrient quality and quantity affected growth patterns, as cultures established on Gamborg B5 medium had comparatively long exponential phase compared to Murashige and Skoog medium, while growth was slow but steady until last phases of the culture on Schenk and Hildebrandt medium. Similarly, we observed that boron deficiency and nitrogen limitation in culture medium (Gamborg B5 medium) enhanced callus biomass (fresh weight 413 g/l and dry weight 20.7 g/l), phenolics production (667.60 mg/l), and lignan content (secoisolariciresinol diglucoside 6.33 and lariciresinol diglucoside 5.22 mg/g dry weight respectively) at 16/8 h light and dark-week 4, while that of neolignans (dehydrodiconiferyl alcohol glucoside 44.42 and guaiacylglycerol-β-coniferyl alcohol ether glucoside 9.26 mg/g dry weight, respectively) in continuous dark after 4th week of culture. Conversely, maximum flavonoids production occurred at both Murashige and Skoog, Schenk and Hildebrandt media (both media types contain comparatively higher boron and nitrogen content) in the presence of continuous light. Generally, continuous dark had no significant role in any growth associated parameter. This study opens new dimension for optimizing growing conditions and evaluating underlying mechanisms in biosynthesis of lignans and neolignans in in vitro cultures of Linum usitatissimum.

Topics: Biomass; Boron; Butylene Glycols; Chromatography, High Pressure Liquid; Flavonoids; Flax; Free Radical Scavengers; Glucosides; Kinetics; Light; Lignans; Nitrogen; Phenols

This study evaluated the protective effect of flaxseed oil (FO) and flaxseed lignan secoisolariciresinol diglucoside (SDG) against oxidative stress in rats with metabolic syndrome (MS). 48 rats were allocated into the following 6 groups: Groups 1 (control), 5 (FO), and 6 (SDG) received water and were treated daily orally with saline, FO, and SDG, respectively. Groups 2 (MS), 3 (MS+FO), and 4 (MS+SDG) received 30% fructose in drinking water for MS induction and were treated daily orally with saline, FO, and SDG, respectively. After 30 d, animals were sacrificed, and blood was collected for biochemical and oxidative analysis. Body weight was recorded weekly. Systolic blood pressure (SBP) was measured before and after treatment. Fructose could produce MS and oxidative stress. FO and SDG prevented changes in SBP, lipids, and glucose. FO and SDG prevented oxidative damage to lipids, and only FO prevented oxidative damage to proteins associated to MS. FO and SDG improved enzymatic antioxidants defenses and reduced glutathione levels, which was greater with SDG. Total polyphenol levels were enhanced in groups that received SDG. Thus, the results of this study demonstrated that treatment with a 30% fructose solution for 30 d is effective for MS induction and the oxidative stress is involved in the pathophysiology of MS induced by fructose-rich diets. Furthermore, we demonstrated that the antioxidant effects attributed to flaxseed are mainly due to its high lignan content especially that of SDG, suggesting that this compound can be used in isolation to prevent oxidative stress associated with MS.. We report that the antioxidant effects attributed to flaxseed are mainly due to its high lignan content, especially that of secoisolariciresinol diglucoside. This is significant because suggests that this compound can be used in isolation to prevent oxidative stress associated with MS. Furthermore, this study was the only one to perform a comparison of the abilities of 2 components of flaxseed to protect against oxidative stress in an MS model, which brings a great advance in the medicine's field, since it indicates another alternative for improve the health and the quality of life of patients with this disorder.

Topics: Animals; Antioxidants; Body Weight; Butylene Glycols; Flax; Fructose; Glucose; Glucosides; Humans; Lignans; Linseed Oil; Lipids; Male; Metabolic Syndrome; Oxidative Stress; Protective Agents; Quality of Life; Rats

The main flax lignan, secoisolariciresinol diglucoside, is stored in a macromolecule containing other ester-bound phenolic compounds. In this study, NMR and HPLC-UV analyses were performed on flaxseeds harvested at different developmental stages to identify and quantify the main phenolic compounds produced during seed development. Extraction was carried out with or without alkaline hydrolysis to determine if these molecules accumulate in the lignan macromolecule and/or in a free form. Monolignol glucosides accumulate in a free form up to 9.85mg/g dry matter at the early developmental stages. Hydroxycinnamic acid glucosides and flavonoid accumulate (up to 3.18 and 4.07mg/g dry matter, respectively) in the later developmental stages and are ester-bound in the lignan macromolecule. Secosiolariciresinol diglucoside accumulates (up to 28.65mg/g dry matter) in the later developmental stages in both forms, mainly ester-bound in the lignan macromolecule and slightly in a free form.

Topics: Butylene Glycols; Flavonoids; Flax; Glucosides; Hydrolysis; Kinetics; Lignans; Macromolecular Substances; Magnetic Resonance Spectroscopy; Phenols; Seeds

Flax lignan, commonly known as secoisolariciresinol (SECO) diglucoside (SDG), has recently been reported with health-promoting activities, including its positive impact in metabolic diseases. However, not much was reported on the biosynthesis of SDG and its monoglucoside (SMG) until lately. Flax UGT74S1 was recently reported to sequentially glucosylate SECO into SMG and SDG in vitro. However, whether this gene is the only UGT achieving SECO glucosylation in flax was not known.. Flax genome-wide mining for UGTs was performed. Phylogenetic and gene duplication analyses, heterologous gene expression and enzyme assays were conducted to identify family members closely related to UGT74S1 and to establish their roles in SECO glucosylation. A total of 299 different UGTs were identified, of which 241 (81%) were duplicated. Flax UGTs diverged 2.4-153.6 MYA and 71% were found to be under purifying selection pressure. UGT74S1, a single copy gene located on chromosome 7, displayed no evidence of duplication and was deemed to be under positive selection pressure. The phylogenetic analysis identified four main clusters where cluster 4, which included UGT74S1, was the most diverse. The duplicated UGT74S4 and UGT74S3, located on chromosomes 8 and 14, respectively, were the most closely related to UGT74S1 and were differentially expressed in different tissues. Heterologous expression levels of UGT74S1, UGT74S4 and UGT74S3 proteins were similar but UGT74S4 and UGT74S3 glucosylation activity towards SECO was seven fold less than UGT74S1. In addition, they both failed to produce SDG, suggesting neofunctionalization following their divergence from UGT74S1.. We showed that UGT74S1 is closely related to two duplicated genes, UGT74S4 and UGT74S3 which, unlike UGT74S1, failed to glucosylate SMG into SDG. The study suggests that UGT74S1 may be the key player in controlling SECO glucosylation into SDG in flax although its closely related genes may also contribute to a minor extent in supplying the SMG precursor to UGT74S1.

Topics: Butylene Glycols; Evolution, Molecular; Flax; Gene Duplication; Gene Expression; Genes, Plant; Genetic Variation; Genome, Plant; Glucosides; Lignans

Malignant mesothelioma (MM), linked to asbestos exposure, is a highly lethal form of thoracic cancer with a long latency period, high mortality and poor treatment options. Chronic inflammation and oxidative tissue damage caused by asbestos fibers are linked to MM development. Flaxseed lignans, enriched in secoisolariciresinol diglucoside (SDG), have antioxidant, anti-inflammatory and cancer chemopreventive properties. As a prelude to chronic chemoprevention studies for MM development, we tested the ability of flaxseed lignan component (FLC) to prevent acute asbestos-induced inflammation in MM-prone Nf2(+/mu) mice. Mice (n = 16-17 per group) were placed on control (CTL) or FLC-supplemented diets initiated 7 days prior to a single intraperitoneal bolus of 400 µg of crocidolite asbestos. Three days post asbestos exposure, mice were evaluated for abdominal inflammation, proinflammatory/profibrogenic cytokine release, WBC gene expression changes and oxidative and nitrosative stress in peritoneal lavage fluid (PLF). Asbestos-exposed mice fed CTL diet developed acute inflammation, with significant (P < 0.0001) elevations in WBCs and proinflammatory/profibrogenic cytokines (IL-1ß, IL-6, TNFα, HMGB1 and active TGFß1) relative to baseline (BL) levels. Alternatively, asbestos-exposed FLC-fed mice had a significant (P < 0.0001) decrease in PLF WBCs and proinflammatory/profibrogenic cytokine levels relative to CTL-fed mice. Importantly, PLF WBC gene expression of cytokines (IL-1ß, IL-6, TNFα, HMGB1 and TGFß1) and cytokine receptors (TNFαR1 and TGFßR1) were also downregulated by FLC. FLC also significantly (P < 0.0001) blunted asbestos-induced nitrosative and oxidative stress. FLC reduces acute asbestos-induced peritoneal inflammation, nitrosative and oxidative stress and may thus prove to be a promising agent in the chemoprevention of MM.

Topics: Animals; Antioxidants; Asbestos, Crocidolite; Butylene Glycols; Chromatography, Liquid; Diet; Dietary Supplements; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Flax; Glucosides; Inflammation; Lignans; Mesothelioma; Mice; Mice, Mutant Strains; Oxidative Stress; Peritoneal Lavage; Peritoneum; Precancerous Conditions; Reverse Transcriptase Polymerase Chain Reaction; Seeds; Tandem Mass Spectrometry; Transcriptome

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

Secoisolariciresinol diglucoside (SDG), the main lignan in whole grain flaxseed, is a potent antioxidant and free radical scavenger with known radioprotective properties. However, the exact mechanism of SDG radioprotection is not well understood. The current study identified a novel mechanism of DNA radioprotection by SDG in physiological solutions by scavenging active chlorine species (ACS) and reducing chlorinated nucleobases.. The ACS scavenging activity of SDG was determined using two highly specific fluoroprobes: hypochlorite-specific 3'-(p-aminophenyl) fluorescein (APF) and hydroxyl radical-sensitive 3'-(p-hydroxyphenyl) fluorescein (HPF). Dopamine, an SDG structural analog, was used for proton (1)H NMR studies to trap primary ACS radicals. Taurine N-chlorination was determined to demonstrate radiation-induced generation of hypochlorite, a secondary ACS. DNA protection was assessed by determining the extent of DNA fragmentation and plasmid DNA relaxation following exposure to ClO(-) and radiation. Purine base chlorination by ClO(-) and γ-radiation was determined by using 2-aminopurine (2-AP), a fluorescent analog of 6-aminopurine.. Chloride anions (Cl(-)) consumed >90% of hydroxyl radicals in physiological solutions produced by γ-radiation resulting in ACS formation, which was detected by (1)H NMR. Importantly, SDG scavenged hypochlorite- and γ-radiation-induced ACS. In addition, SDG blunted ACS-induced fragmentation of calf thymus DNA and plasmid DNA relaxation. SDG treatment before or after ACS exposure decreased the ClO(-) or γ-radiation-induced chlorination of 2-AP. Exposure to γ-radiation resulted in increased taurine chlorination, indicative of ClO(-) generation. NMR studies revealed formation of primary ACS radicals (chlorine atoms (Cl) and dichloro radical anions (Cl2¯)), which were trapped by SDG and its structural analog dopamine.. We demonstrate that γ-radiation induces the generation of ACS in physiological solutions. SDG treatment scavenged ACS and prevented ACS-induced DNA damage and chlorination of 2-aminopurine. This study identified a novel and unique mechanism of SDG radioprotection, through ACS scavenging, and supports the potential usefulness of SDG as a radioprotector and mitigator for radiation exposure as part of cancer therapy or accidental exposure.

Topics: 2-Aminopurine; Animals; Antioxidants; Butylene Glycols; Cattle; Chlorine; DNA; DNA Fragmentation; Flax; Free Radical Scavengers; Gamma Rays; Glucosides; Hydroxyl Radical; Lignans; Lipid Peroxidation; Plasmids; Radiation-Protective Agents

Secoisolariciresinol diglucoside (SDG), a lignan extracted from flaxseed, has been shown to suppress benign prostatic hyperplasia (BPH). However, little is known about the mechanistic basis for its anti-BPH activity. The present study showed that enterolactone (ENL), the mammalian metabolite of SDG, shared the similar binding site of G1 on a new type of membranous estrogen receptor, G-protein-coupled estrogen eceptor 1 (GPER), by docking simulations method. ENL and G1 (the specific agonist of GPER) inhibited the proliferation of human prostate stromal cell line WPMY-1 as shown by MTT assay and arrested cell cycle at the G0/G1 phase, which was displayed by propidium iodide staining following flow cytometer examination. Silencing GPER by short interfering RNA attenuated the inhibitory effect of ENL on WPMY-1 cells. The therapeutic potential of SDG in the treatment of BPH was confirmed in a testosterone propionate-induced BPH rat model. SDG significantly reduced the enlargement of the rat prostate and the number of papillary projections of prostatic alveolus and thickness of the pseudostratified epithelial and stromal cells when comparing with the model group. Mechanistic studies showed that SDG and ENL increased the expression of GPER both in vitro and in vivo. Furthermore, ENL-induced cell cycle arrest may be mediated by the activation of GPER/ERK pathway and subsequent upregulation of p53 and p21 and downregulation of cyclin D1. This work, in tandem with previous studies, will enhance our knowledge regarding the mechanism(s) of dietary phytochemicals on BPH prevention and ultimately expand the scope of adopting alternative approaches in BPH treatment.

Topics: 4-Butyrolactone; Animals; Antineoplastic Agents, Phytogenic; Binding Sites; Butylene Glycols; Cell Line, Tumor; Cell Proliferation; Dietary Supplements; Flax; Gene Expression Regulation, Neoplastic; Glucosides; Glycosides; Humans; Lignans; Male; Models, Molecular; Molecular Docking Simulation; Neoplasm Proteins; Prostate; Prostatic Hyperplasia; Random Allocation; Rats; Rats, Wistar; Receptors, Estrogen; Receptors, G-Protein-Coupled; RNA Interference; Seeds

Topics: Amygdalin; Bread; Butylene Glycols; Chromatography, High Pressure Liquid; Flax; Flour; Food, Fortified; Glucosides; Glutens; Glycosides; Lignans; Magnetic Resonance Spectroscopy; Nitriles; Temperature

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

An integrated omics approach using genomics, transcriptomics, metabolomics (MALDI mass spectrometry imaging, MSI), and bioinformatics was employed to study spatiotemporal formation and deposition of health-protecting polymeric lignans and plant defense cyanogenic glucosides. Intact flax (Linum usitatissimum) capsules and seed tissues at different development stages were analyzed. Transcriptome analyses indicated distinct expression patterns of dirigent protein (DP) gene family members encoding (-)- and (+)-pinoresinol-forming DPs and their associated downstream metabolic processes, respectively, with the former expressed at early seed coat development stages. Genes encoding (+)-pinoresinol-forming DPs were, in contrast, expressed at later development stages. Recombinant DP expression and DP assays also unequivocally established their distinct stereoselective biochemical functions. Using MALDI MSI and ion mobility separation analyses, the pinoresinol downstream derivatives, secoisolariciresinol diglucoside (SDG) and SDG hydroxymethylglutaryl ester, were localized and detectable only in early seed coat development stages. SDG derivatives were then converted into higher molecular weight phenolics during seed coat maturation. By contrast, the plant defense cyanogenic glucosides, the monoglucosides linamarin/lotaustralin, were detected throughout the flax capsule, whereas diglucosides linustatin/neolinustatin only accumulated in endosperm and embryo tissues. A putative biosynthetic pathway to the cyanogens is proposed on the basis of transcriptome coexpression data. Localization of all metabolites was at ca. 20 μm resolution, with the web based tool OpenMSI enabling not only resolution enhancement but also an interactive system for real-time searching for any ion in the tissue under analysis.

Topics: Butylene Glycols; Flax; Furans; Glucosides; Glycosides; Lignans; Molecular Structure; Nitriles; Seeds; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Previously, from the human intestinal flora we isolated the bacterial strain Bacteroides uniformis ZL1, which could convert secoisolariciresinol diglucoside (SDG) to its aglycone secoisolariciresinol (SECO) in vivo. In this study, 24 putative β-glucosidase genes were screened from the genome of B. uniformis ATCC 8492, which were used as templates to design PCR primers for the target genes in B. uniformis ZL1. Fifteen genes (bgl1-bgl15) were amplified from strain ZL1, and among them we identified bgl8 as the gene encoding the SDG-hydrolyzing β-glucosidase. We sequenced the bgl8 gene, cloned it into the expression vector and then transformed Escherichia coli to construct the recombinant bacteria that could synthesize the target β-glucosidase (BuBGL8). We purified and characterized BuBGL8, which showed maximal activity and stability under the culture conditions of pH 6.0 and 30 °C. SDG (2.0 mg/ml) was converted to SECO by both the purified BuBGL8 (0.035 mg/ml) and crude enzyme extract (0.23 mg crude protein/ml) with the efficiency of more than 90 % after 90 min at the reaction conditions. This is, to our knowledge, the first report of using recombinant bacteria to synthesize the SDG-hydrolyzing β-glucosidase, which could be used to produce SECO from SDG conveniently and highly efficiently.

Topics: Amino Acid Sequence; Bacteroides; beta-Glucosidase; Butylene Glycols; Cloning, Molecular; DNA, Bacterial; Enzyme Stability; Escherichia coli; Gene Expression; Glucosides; Hydrogen-Ion Concentration; Hydrolysis; Lignans; Models, Molecular; Molecular Docking Simulation; Molecular Sequence Data; Recombinant Proteins; Sequence Alignment; Sequence Analysis, DNA; Temperature

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

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

Lignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta. Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans. Glycosylation is a process by which a glycosyl group is covalently attached to an aglycone substrate and is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Until now, very little information was available on UGT genes that may play a role in flax SDG biosynthesis. Here we report on the identification, structural and functional characterization of 5 putative UGTs potentially involved in secoisolariciresinol (SECO) glucosylation in flax.. Five UGT genes belonging to the glycosyltransferases' family 1 (EC 2.4.x.y) were cloned and characterized. They fall under four UGT families corresponding to five sub-families referred to as UGT74S1, UGT74T1, UGT89B3, UGT94H1, UGT712B1 that all display the characteristic plant secondary product glycosyltransferase (PSPG) conserved motif. However, diversity was observed within this 44 amino acid sequence, especially in the two peptide sequences WAPQV and HCGWNS known to play a key role in the recognition and binding of diverse aglycone substrates and in the sugar donor specificity. In developing flax seeds, UGT74S1 and UGT94H1 showed a coordinated gene expression with that of pinoresinol-lariciresinol reductase (PLR) and their gene expression patterns correlated with SDG biosynthesis. Enzyme assays of the five heterologously expressed UGTs identified UGT74S1 as the only one using SECO as substrate, forming SECO monoglucoside (SMG) and then SDG in a sequential manner.. We have cloned and characterized five flax UGTs and provided evidence that UGT74S1 uses SECO as substrate to form SDG in vitro. This study allowed us to propose a model for the missing step in SDG lignan biosynthesis.

Topics: Amino Acid Motifs; Amino Acid Sequence; Biosynthetic Pathways; Butylene Glycols; Chromatography, High Pressure Liquid; Cloning, Molecular; Conserved Sequence; DNA, Complementary; Enzyme Assays; Expressed Sequence Tags; Flax; Gene Expression Profiling; Gene Expression Regulation, Plant; Genes, Plant; Glucosides; Glycosylation; Glycosyltransferases; Kinetics; Lignans; Mass Spectrometry; Molecular Sequence Data; Organ Specificity; Oxidoreductases; Phylogeny; Sesamum

Wholegrain flaxseed (FS), and its lignan component (FLC) consisting mainly of secoisolariciresinol diglucoside (SDG), have potent lung radioprotective properties while not abrogating the efficacy of radiotherapy. However, while the whole grain was recently shown to also have potent mitigating properties in a thoracic radiation pneumonopathy model, the bioactive component in the grain responsible for the mitigation of lung damage was never identified. Lungs may be exposed to radiation therapeutically for thoracic malignancies or incidentally following detonation of a radiological dispersion device. This could potentially lead to pulmonary inflammation, oxidative tissue injury, and fibrosis. This study aimed to evaluate the radiation mitigating effects of FLC in a mouse model of radiation pneumonopathy.. We evaluated FLC-supplemented diets containing SDG lignan levels comparable to those in 10% and 20% whole grain diets. 10% or 20% FLC diets as compared to an isocaloric control diet (0% FLC) were given to mice (C57/BL6) (n=15-30 mice/group) at 24, 48, or 72-hours after single-dose (13.5 Gy) thoracic x-ray treatment (XRT). Mice were evaluated 4 months post-XRT for blood oxygenation, lung inflammation, fibrosis, cytokine and oxidative damage levels, and survival.. FLC significantly mitigated radiation-related animal death. Specifically, mice fed 0% FLC demonstrated 36.7% survival 4 months post-XRT compared to 60-73.3% survival in mice fed 10%-20% FLC initiated 24-72 hours post-XRT. FLC also mitigated radiation-induced lung fibrosis whereby 10% FLC initiated 24-hours post-XRT significantly decreased fibrosis as compared to mice fed control diet while the corresponding TGF-beta1 levels detected immunohistochemically were also decreased. Additionally, 10-20% FLC initiated at any time point post radiation exposure, mitigated radiation-induced lung injury evidenced by decreased bronchoalveolar lavage (BAL) protein and inflammatory cytokine/chemokine release at 16 weeks post-XRT. Importantly, neutrophilic and overall inflammatory cell infiltrate in airways and levels of nitrotyrosine and malondialdehyde (protein and lipid oxidation, respectively) were also mitigated by the lignan diet.. Dietary FLC given early post-XRT mitigated radiation effects by decreasing inflammation, lung injury and eventual fibrosis while improving survival. FLC may be a useful agent, mitigating adverse effects of radiation in individuals exposed to incidental radiation, inhaled radioisotopes or even after the initiation of radiation therapy to treat malignancy.

Topics: Animal Feed; Animals; Bronchoalveolar Lavage Fluid; Butylene Glycols; Cytokines; Female; Fibrosis; Flax; Glucosides; Kaplan-Meier Estimate; Lignans; Lung; Lung Injury; Malondialdehyde; Mice; Mice, Inbred C57BL; Neutrophils; Oxygen; Phytotherapy; Radiation Injuries, Experimental; Radiation Pneumonitis; Radiation-Protective Agents; Seeds; Survival Rate; Time Factors; Transforming Growth Factor beta1; Tyrosine

Flaxseed (FS) has a breast tumor-reducing effect, possibly because of its high content of secoisolariciresinol diglucoside (SDG) lignan. Sesame seed (SS) is rich in the lignan sesamin (SES) but is non-protective. Both lignans are metabolized to estrogen-like enterodiol and enterolactone. The objective of this study was to differentiate the effects of SDG and SES on established human estrogen receptor-positive breast tumors (MCF-7) in athymic mice with high serum estrogen to help explain the different effects of FS and SS. Mice were fed for 8 wk the basal diet (BD, control) or BD supplemented with 1 g/kg SDG or SES. SES reduced palpable tumor size by 23% compared to control, whereas SDG did not differ from SES or control. Both treatments reduced tumor cell proliferation, but only SES increased apoptosis. SDG and SES reduced human epidermal growth factor receptor 2 and endothelial growth factor receptor expressions, but only SES reduced downstream pMAPK. Neither treatment affected IGF-1R, vascular endothelial growth factor receptor-2, Akt, pAkt, or MAPK of the growth factor signaling pathway. Thus, at high serum estrogen levels, SDG may not account for the tumor reducing effect of FS. SES was more effective than SDG in reducing breast tumor growth, but its effect may have been lost when consumed as a component of SS.

Topics: Animals; Apoptosis; Body Weight; Breast Neoplasms; Butylene Glycols; Cell Line, Tumor; Cell Proliferation; Dioxoles; Eating; Estrogens; Female; Flax; Glucosides; Humans; Lignans; Mice; Mice, Nude; Receptor, ErbB-2; Receptor, IGF Type 1; Receptors, Estrogen; Seeds; Sesamum; Signal Transduction; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays

Mature seeds of 20 Linum species were analyzed for their content of lignans. The seeds of common flax (Linum usitatissimum L.) are known to contain as characteristic lignan sesoisolariciresinol diglucoside (SDG), whose presence in seeds of some other Linum species has also been reported. In order to investigate the material for the presence of such very polar lignans as well as for less polar non-glycosidic lignans as frequently found in aerial parts of Linum species, polar and non-polar extracts of each sample were analyzed by HPLC/ESI-MSMS. SDG was detected in 15 of 16 investigated seed samples of taxa representing sections Linum and Dasylinum. None of eight samples of taxa from sections Syllinum and Linopsis contained detectable amounts of SDG. Quite interestingly, most of the SDG-positive samples contained the 8R,8'R-isomer exclusively while only three (including L. usitatissimum) contained the 8S,8'S-stereoisomer as the predominant form. As a most noteworthy finding, the dichloromethane extracts obtained from seeds of several Linum species were found to contain significant concentrations of non-polar cyclolignans of the arylnaphthalene/-dihydronaphthalene lactone type or, alternatively of the aryltetralin lactone type. Thus, seeds of Linum perenne L. as well as those of several other representatives of sections Linum and Dasylinum were found to contain significant concentrations of the arylnaphthalene justicidin B along with further compounds of this type and some aryldihydronaphthalene-type lignans. On the other hand, seeds of Linum flavum and further representatives of section Syllinum were found to contain aryltetralin-type lignans, mainly in the form of esters with aliphatic carboxylic acids, such as 6-methoxypodophyllotoxin-7-O-n-hexanoate, whose occurrence in L. flavum seeds has very recently been reported by us for the first time. Various chemosystematic and biogenetic aspects are discussed in the light of these results.

Topics: Butylene Glycols; Flax; Glucosides; Hydrolysis; Lignans; Methylene Chloride; Naphthalenes; Seeds

Secoisolariciresinol (SECO) is increasingly recognized for potential clinical application because of its preventive effects against breast and colon cancers, atherosclerosis and diabetes, and its production through biotransformation has been attempted. However, previously reported bacteria all required stringent anaerobic culture conditions, precluding large-scale production. Here, we report the isolation and characterization of bacteria that produce SECO under less stringent anaerobic culture conditions.. Using defatted flaxseed as raw material, we isolated a facultative anaerobic bacterium from human faeces that hydrolysed secoisolariciresinol diglucoside-3-hydroxy-3-methyl glutaric acid (SDG-HMGA) oligomers in flaxseed to produce SECO. Both conventional assays and 16S rRNA gene sequence analysis demonstrated its close relatedness with Bacteroides uniformis. The transformation efficiency of SDG in defatted flaxseed to SECO was more than 80% by this bacterial strain. We investigated factors that might influence fermentation, such as redox potential and pH, for large-scale fermentation of defatted flaxseed to produce SECO.. The method to produce SECO through biotransformation of defatted flaxseed with this bacterial strain is highly efficient and economic.. This bacterial strain can transform SDG to SECO under less stringent anaerobic culture conditions, which will greatly facilitate industry-scale production of SECO.

Topics: Adult; Bacteroidaceae; Biotransformation; Butylene Glycols; Chromatography, High Pressure Liquid; Feces; Female; Fermentation; Flax; Glucosides; Humans; Hydrolysis; Lignans; Male; Phylogeny; RNA, Ribosomal, 16S; Young Adult

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

While dietary wholegrain Flaxseed (FS) has potent anti-inflammatory, anti-fibrotic and antioxidant properties in murine models of acute and chronic lung injury, the main bioactive ingredient that contributes to these protective effects remains unknown. This study evaluated the lignan complex of FS (FLC) enriched in secoisolariciresinol diglucoside with respect to lung radioprotective and tumor radiosensitizing efficacy using a mouse model of thoracic radiation-induced pneumonopathy. C57/Bl6 mice were fed 0% FS, 10% FS, 10% FLC or 20% FLC for 3 weeks, then irradiated with a single fraction (13.5 Gy) of X-ray radiation treatment (XRT). Mouse survival was monitored for 4 months after irradiation and inflammatory lung parameters were evaluated in bronchoalveolar lavage (BAL) fluid. Gene and protein levels of protective antioxidant and phase II enzymes were evaluated in lung tissue using qPCR and protein levels were verified by immunoblotting. Prolonged administration of the FLC diet was well tolerated and was not associated with any toxicity. Importantly, comparable to the whole grain 10% FS diet, irradiated mice fed 10% and 20% FLC diets displayed improved survival. Improved hemodynamic measurements were also recorded in irradiated mice fed 10% FS or 10% FLC diet compared to irradiated 0% FS fed mice. Flaxseed lignan complex diet also attenuated polymorphonuclear infiltration and overall lung inflammation to levels comparable to those in nonirradiated mice. Flaxseed lignan complex, similarly to FS, up-regulated gene expression as well as protein levels of protective antioxidant enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). Dietary FLC induced radiosensitizing effects in our murine model of metastatic lung cancer. Importantly, protection of normal tissue does not thwart tumor cell death by radiation treatment. The dietary lignan complex of FS, mainly consisting of the phenolic secoisolariciresinol, is protective against radiation pneumonopathy in vivo while not hindering the tumoricidal effects of radiotherapy.

Topics: Animals; Antioxidants; Butylene Glycols; Dietary Carbohydrates; Eating; Female; Flax; Glucosides; Inflammation; Lignans; Lung; Mice; Mice, Inbred C57BL; Phenols; Radiation Pneumonitis; Radiation-Protective Agents; Survival Analysis; Thorax; Weight Loss

Nutritional value is a priority in new product development. Using vegetable or marine oils, rich in polyunsaturated fatty acids, in dairy beverage formulations is an option to provide the consumers with healthier products. However, these formulations are prone to oxidation, which is responsible for rapid flavour degradation and the development of potentially toxic reaction products during storage. Flaxseed lignans, secoisolariciresinol diglucoside (SDG), and its mammalian metabolites have antioxidant activity and could be used in beverage formulations to prevent oxidation. Commercially available SDG extract was added to the formulation of dairy beverages enriched with flaxseed oil. As an alternative approach, dairy beverages were produced from milk naturally rich in SDG metabolites obtained through the alteration of cow diet. Resistance to oxidation was determined from the kinetics of hexanal and propanal production during heat and light exposure treatments. Increasing SDG concentration in dairy beverage slightly reduced redox potential but had no effect on oxygen consumption during oxidation treatments. The presence of SDG in dairy beverage significantly improved resistance to heat- and light-induced oxidation. However, purified enterolactone, a mammalian metabolite from SDG, prevented oxidation at much lower concentrations. The use of milk from dairy cow fed flaxseed meal did not improve resistance to oxidation in dairy beverage. Enterolactone concentration in milk was increased by the experimental diet but it remained too low to observe any significant effect on dairy beverage oxidation.

Topics: 4-Butyrolactone; Animals; Antioxidants; Butylene Glycols; Cattle; Diet; Fatty Acids, Unsaturated; Female; Flax; Food, Fortified; Glucosides; Hot Temperature; Lignans; Milk; Oxidation-Reduction; Oxidative Stress; Photochemical Processes

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

This study aimed to elucidate which component of flaxseed, i.e. secoisolariciresinol diglucoside (SDG) lignan or flaxseed oil (FO), makes tamoxifen (TAM) more effective in reducing growth of established estrogen receptor positive breast tumors (MCF-7) at low circulating estrogen levels, and potential mechanisms of action. In a 2 x 2 factorial design, ovariectomized athymic mice with established tumors were treated for 8 wk with TAM together with basal diet (control), or basal diet supplemented with SDG (1 g/kg diet), FO (38.5 g/kg diet), or combined SDG and FO. SDG and FO were at levels in 10% flaxseed diet. Palpable tumors were monitored and after animal sacrifice, analyzed for cell proliferation, apoptosis, ER-mediated (ER-alpha, ER-beta, trefoil factor 1, cyclin D1, progesterone receptor, AIBI), growth factor-mediated (epidermal growth factor receptor, human epidermal growth factor receptor-2, insulin-like growth factor receptor-1, phosphorylated mitogen activated protein kinase, PAKT, BCL2) signaling pathways and angiogenesis (vascular endothelial growth factor). All treatments reduced the growth of TAM-treated tumors by reducing cell proliferation, expression of genes, and proteins involved in the ER- and growth factor-mediated signaling pathways with FO having the greatest effect in increasing apoptosis compared with TAM treatment alone. SDG and FO reduced the growth of TAM-treated tumors but FO was more effective. The mechanisms involve both the ER- and growth factor-signaling pathways.

Topics: Animals; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Butylene Glycols; Cell Line, Tumor; Cell Proliferation; Female; Flax; Gene Expression Regulation, Neoplastic; Glucosides; Humans; Lignans; Linseed Oil; Mice; Mice, Nude; Phytotherapy; Random Allocation; Receptors, Estrogen; Receptors, Growth Factor; RNA, Messenger; Seeds; Signal Transduction; Tamoxifen; Tumor Burden; Xenograft Model Antitumor Assays

Our previous study demonstrated that lignan metabolites enterolactone and enterodiol inhibited colonic cancer cell growth by inducing cell cycle arrest and apoptosis. However, the dietary lignans are naturally present as glycoside precursors, such as secoisolariciresinol diglucoside (SDG), which have not been evaluated yet. This study tested the hypothesis that dietary SDG might have a different effect than its metabolites in human colonic SW480 cancer cells. Treatment with SDG at 0 to 40 μmol/L for up to 48 hours resulted in a dose- and time-dependent decrease in cell numbers, which was comparable to enterolactone. The inhibition of cell growth by SDG did not appear to be mediated by cytotoxicity, but by a cytostatic mechanism associated with an increase of cyclin A expression. Furthermore, high-performance liquid chromatography analysis indicated that SDG in the media was much more stable than enterolactone (95% of SDG survival vs 57% of enterolactone after 48-hour treatment). When the cells were treated with either enterolactone or SDG at 40 μmol/L for 48 hours, the intracellular levels of enterolactone, as measured by high-performance liquid chromatography-mass spectrometry/electron spray ionization, were about 8.3 × 10(-8) nmol per cell; but intracellular SDG or potential metabolites were undetectable. Taken together, SDG demonstrated similar effects on cell growth, cytotoxicity, and cell cycle arrest when compared with its metabolite enterolactone. However, the reliable stability and undetectable intracellular SDG in treated cells may suggest that metabolism of SDG, if exposed directly to the colonic cells, could be different from the known degradation by microorganisms in human gut.

Topics: 4-Butyrolactone; Adenocarcinoma; Anticarcinogenic Agents; Butylene Glycols; Cell Cycle; Cell Line, Tumor; Chromatography, High Pressure Liquid; Colonic Neoplasms; Cyclin A; Cytostatic Agents; Dietary Carbohydrates; Dose-Response Relationship, Drug; Glucosides; Glycosides; Humans; Lignans; Mass Spectrometry

A new method of flaxseed-derived lignan determination was developed using HPLC with high-resolution time-of-flight MS (TOF-MS), optimized, and compared to two existing methods (HPLC/MS/MS and GC/MS). The limits of detection (LODs) for HPLC/TOF-MS (0.002-0.043 pg) were comparable with those of the optimized and improved HPLC/MS/MS (0.001-0.015 pg), whereas the LODs for the optimized GC/MS were higher (0.02-3.0 pg, yet lower than reported before). Using the newly developed detection and separation methods, several key flaxseed sample preparation parameters (including extraction, hydrolysis, and sample purification) were evaluated resulting in the development of efficient protocol for lignan quantification from flaxseed hulls and embryos. The results confirmed the importance of quantification of both aglycones and unhydrolyzed glucosides in order to obtain the total lignan estimates.

Topics: Antioxidants; Butylene Glycols; Chemical Fractionation; Chromatography, High Pressure Liquid; Chromatography, Liquid; Flax; Gas Chromatography-Mass Spectrometry; Glucosides; Least-Squares Analysis; Lignans; Mass Spectrometry; Models, Chemical; Reproducibility of Results; Sensitivity and Specificity; Solid Phase Microextraction; Tandem Mass Spectrometry

The present study involved a comparative analysis of the effects of purified flaxseed lignans, secoisolariciresinol diglucoside (SDG) and its aglycone metabolite (SECO), in hyperlipidaemic rats. For hypercholesterolaemia, female Wistars (six rats per group) were fed a standard or 1 % cholesterol diet and orally administered 0, 3 or 6 mg SDG/kg or 0, 1.6 or 3.2 mg SECO/kg body weight once daily for 4 weeks. Hypertriacylglycerolaemia was induced in male Sprague-Dawley rats (ten rats per group) by supplementing tap water with 10 % fructose. These rats were orally administered 0, 3 or 6 mg SDG/kg body weight once daily for 2 weeks. Fasting blood samples (12 h) were collected predose and at the end of the dosing period for serum lipid analyses. Rats were killed and livers rapidly excised and sectioned for lipid, mRNA and histological analyses. Chronic administration of equimolar amounts of SDG and SECO caused similar dose-dependent reductions in rate of body-weight gain and in serum total and LDL-cholesterol levels and hepatic lipid accumulation. SDG and SECO failed to alter hepatic gene expression of commonly reported regulatory targets of lipid homeostasis. SDG had no effect on serum TAG, NEFA, phospholipids and rate of weight gain in 10 % fructose-supplemented rats. In conclusion, our data suggest that the lignan component of flaxseed contributes to the hypocholesterolaemic effects of flaxseed consumption observed in humans. Future studies plan to identify the biochemical mechanism(s) through which flaxseed lignans exert their beneficial effects and the lignan form(s) responsible.

Topics: Animals; Base Sequence; Body Weight; Butylene Glycols; Dose-Response Relationship, Drug; Female; Flax; Fructose; Gene Expression; Glucosides; Hyperlipidemias; Lignans; Lipids; Liver; Male; Models, Animal; Molecular Sequence Data; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar

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

A human intestinal bacterium Ruminococcus (R.) sp. END-1 capable of oxidizing (-)-enterodiol to (-)-enterolactone, enantioselectively, was further investigated from the perspective of transformation of plant lignans to mammalian lignans; A cell-free extract of the bacterium transformed (-)-enterodiol to (-)-enterolactone through an intermediate, enterolactol. The bacterium showed not only oxidation but also demethylation and deglucosylation activities for plant lignans. Arctiin and secoisolariciresinol diglucoside were converted to (-)-dihydroxyenterolactone and (+)-dihydroxyenterodiol, respectively. Moreover, by coincubation with Eggerthella sp. SDG-2, the bacterium transformed arctiin and secoisolariciresinol diglucoside to (-)-enterolactone and (+)-enterodiol, respectively.

Topics: Biotransformation; Butylene Glycols; Furans; Glucosides; Humans; Intestines; Lignans; Molecular Structure; Ruminococcus; Stereoisomerism

Zinc is essential for cell growth. Previous studies have shown that zinc concentration in breast cancer tissues is higher than that in normal breast tissues. Zinc cannot passively diffuse across cell membranes and specific zinc transporter proteins are required. Two gene families have been identified involved in zinc homeostasis. ZnT transporters reduce intracellular zinc while ZIP transporters increase intracellular zinc. In this study, three human zinc transporter members: ZnT-1, ZIP2 and LIV-1 were chosen. We aimed to determine the effect of flaxseed lignan on the growth of ER-negative breast cancer cells in a nude mice model and observe the effect of flaxseed lignan on the regulation of the three zinc transporter in mRNA level. Nude mice were xenografted with human breast cancer cell line MDA-MB-231 and 6 weeks later were fed either the basal diet (BD) or BD supplemented with 10% FS and SDG for 5 weeks. The SDG levels were equivalent to the amounts in the 10% FS. RT-PCR was performed. Compared with the BD group, the tumor growth rate was significantly lower (P < 0. 05) in the FS and SDG group. ZnT-1 mRNA level in mammary tumor was increased in SDG group and decreased in FS group, but no significant difference was found. Extremely low amplification of ZIP2 from mRNA was detected, with no difference between the treatment groups. LIV-1 mRNA expression of SDG group increases compared with BD group. In FS group, it significantly increases nearly 9 times than that in BD group (P < 0. 005).

Topics: Animals; Breast Neoplasms; Butylene Glycols; Cation Transport Proteins; Cell Line, Tumor; Diet; Female; Flax; Glucosides; Humans; Lignans; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Neoplasm Proteins; RNA, Messenger; Transplantation, Heterologous

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

Estrogenic plant compounds from the human diet such as the lignan secoisolariciresinol diglucoside (SDG, 1) can exert biological activity in the human body upon ingestion and bioactivation to enterodiol (END, 5) and enterolactone (ENL, 6). Bioavailability of lignans is influenced by the food matrix and gut microbial action, of which the latter is subject to a large interindividual variation. In this study, the fate of the lignan precursor SDG, present in the lignan macromolecule of flax seed ( Linum usitatissimum), was determined during an artificial stomach and small intestinal digestion and during metabolism by two different enterolignan phenotypes in a TWINSHIME environment (TWIN Simulator of the Human Intestinal Microbial Ecosystem). The lignan macromolecule acted as a delivery system of SDG in the large intestine. SDG was only hydrolyzed into secoisolariciresinol (SECO, 2) through microbial action in the ascending colon, after which it was bioactivated into enterolignans from the transverse colon onward. Single demethylation was a first step in the bioactivation, followed by dehydroxylation. Enterolignan phenotypes remained stable throughout the experimental period. The establishment of END and ENL production equilibria reflected the subdominance of ENL-producing bacteria in the gastrointestinal tract.

Topics: Bacteria; Butylene Glycols; Diet; Ecosystem; Estrogens; Gastrointestinal Tract; Glucosides; Humans; Lignans; Macromolecular Substances; Models, Biological

Two anaerobic bacteria involved in the conversion of the plant lignan secoisolariciresinol diglucoside were isolated from faeces of a healthy male adult. The first isolate, strain SDG-Mt85-3Db, was a mesophilic strictly anaerobic Gram-positive helically coiled rod. Based on 16S r RNA gene sequence analysis, its nearest relatives were Clostridium cocleatum (96.7% similarity) and Clostridium ramosum (96.6%). In contrast to these species, the isolate was devoid of alpha-galactosidase and -glucosidase and did not grow on maltose, melibiose, raffinose, rhamnose and trehalose. The hypothesis that strain SDG-Mt85-3Db represents a new bacterial species of the Clostridium cluster XVIII was confirmed by DNA-DNA hybridisation experiments. The G+C content of DNA of strain SDG-Mt85-3Db (30.7+/-0.8 mol%) was comparable with that of Clostridium butyricum, the type species of the genus Clostridium. The name Clostridium saccharogumia is proposed for strain SDG-Mt85-3Db (=DSM 17460T=CCUG 51486T). The second isolate, strain ED-Mt61/PYG-s6, was a mesophilic strictly anaerobic Gram-positive regular rod. Based on 16S rRNA gene sequence analysis, its nearest relatives were Clostridium amygdalinum (93.3%), Clostridium saccharolyticum (93.1%) and Ruminococcus productus (93.0%). The isolate differed from these species in its ability to dehydrogenate enterodiol. It also possessed alpha-arabinosidase and -galactosidase and had a higher G+C content of DNA (48.0 mol%). According to these findings, it is proposed to create a novel genus, Lactonifactor, and a novel species, Lactonifactor longoviformis, to accommodate strain ED-Mt61/PYG-s6. The type strain is DSM 17459T (=CCUG 51487T).

Topics: 4-Butyrolactone; Adult; Base Composition; Butylene Glycols; Clostridium; Colon; Culture Media; Dietary Carbohydrates; DNA; DNA, Ribosomal; Feces; Genotype; Glucosides; Gram-Positive Rods; Humans; Lignans; Male; Molecular Sequence Data; Nucleic Acid Hybridization; Phenotype; Phylogeny; Phytoestrogens; RNA, Ribosomal, 16S

Secoisolariciresinol (SECO ) is the major lignan found in flaxseed (Linum usitatissimum L.) and is present in a polymer that contains secoisolariciresinol diglucoside (SDG ). SECO, SDG and the polymer are known to have a number of health benefits, including reduction of serum cholesterol levels, delay in the onset of type II diabetes and decreased formation of breast, prostate and colon cancers. The health benefits of SECO and SDG may be partially attributed to their antioxidant properties. To better understand their antioxidant properties, SECO and SDG were oxidized using 2,2'-azobis(2-amidinopropane), an in vitro model of radical scavenging. The major lignan radical-scavenging oxidation products and their formation over time were determined. SDG was converted to four major products, which were the result of a phenoxyl radical intermediate. One of these products, a dimer of SDG, decomposed under the reaction conditions to form two of the other major products, and . SECO was converted to five major products, two of which were also the result of a phenoxyl radical intermediate. The remaining products were the result of an unexpected alkoxyl radical intermediate. The phenol oxidation products were stable under the reaction conditions, whereas two of the alcohol oxidation products decomposed. In general, only one phenol group on the lignans was oxidized, suggesting that the number of phenols per molecule may not predict radical scavenging antioxidant ability of lignans. Finally, SECO is a superior antioxidant to SDG, and it may be that the additional alcohol oxidation pathway contributes to its greater antioxidant ability.

Topics: Amidines; Antioxidants; Butylene Glycols; Flax; Free Radical Scavengers; Glucosides; Lignans; Molecular Conformation; Oxidation-Reduction; Stereoisomerism; Time Factors

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

The study focused on the effects of processing and storage on the stability of flaxseed-derived secoisolariciresinol diglucoside (SDG) added to various bakery products. The SDG concentration of doughs, baked rye breads, graham buns, and muffins was analyzed by high-performance liquid chromatography-diode array detection; the baked products were analyzed immediately after baking and upon storage at room temperature for 1 week and at -25 degrees C for 1 and 2 months, respectively. Added SDG was found to withstand normal baking temperatures in all bakery products. SDG also was a relatively stable compound during storage. Similarly, the content of SDG in flax buns containing fat-free flaxseed meal was unaffected by storage. We conclude that cereal-based bakery products can be supplemented with flaxseed-derived SDG.

Topics: Butylene Glycols; Drug Stability; Flax; Food Analysis; Food Handling; Food Preservation; Food, Fortified; Glucosides; Lignans

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

Plant lignans occur in many foods, with flaxseed presently recognized as the richest source. Some plant lignans can be converted by intestinal microbiota to the mammalian lignans, enterodiol and enterolactone, which may have protective effects against hormone-related diseases such as breast cancer. This study determined whether plant lignans in sesame seed, particularly sesamin, could be metabolized to the mammalian lignans. The total plant lignan concentration in sesame seed (2180 micromol/100 g) was higher than that in flaxseed (820 micromol/100 g). In vitro fermentation with human fecal inoculum showed conversion of sesamin to the mammalian lignans, although at a lower rate (1.1%) compared with that of secoisolariciresinol diglucoside (57.2%). However, when fed to female Sprague-Dawley rats for 10 d, sesamin (15 mg/kg body weight) and a 10% sesame seed diet resulted in greater (P < 0.05) urinary mammalian lignan excretion (3.2 and 11.2 micromol/d, respectively), than the control (< 0.05 micromol/d). We conclude that sesame seed is a rich source of mammalian lignan precursors and sesamin is one of them. From intermediate metabolites of sesamin identified in rat urine by GC-MS, a tentative metabolic pathway of sesamin to mammalian lignans is suggested.

Topics: Animals; Butylene Glycols; Dioxoles; Feces; Female; Fermentation; Flax; Gas Chromatography-Mass Spectrometry; Glucosides; Humans; Lignans; Mass Spectrometry; Rats; Rats, Sprague-Dawley; Seeds; Sesamum

The human intestinal microbiota is necessary for the production of enterolignans from the dietary lignan secoisolariciresinol diglucoside (SDG). However, little is known about the bacteria that contribute to SDG conversion. Therefore, we aimed at describing the occurrence and activity of SDG metabolising bacteria. The data showed differences in conversion efficiency between SDG deglycosylating species, but SDG was completely deglycosylated within 20 h by five of six strains. The strain Clostridium sp. SDG-Mt85-3Db showed the highest initial rate of SDG deglycosylation. Furthermore, we found that Bacteroides distasonis and B. fragilis made up 0.5% and 3.3% of total faecal bacteria, respectively. However, Clostridium sp. SDG-Mt85-3Db was detected within the dominant microbiota of only two out of 20 faecal samples. Bacteria involved in the demethylation step of SDG conversion also demethylated a variety of compounds other than SDG. In particular, Peptostreptococcus productus demethylated the lignans pinoresinol, lariciresinol and matairesinol. Finally, Eggerthella lenta catalysed the reduction of pinoresinol and lariciresinol to secoisolariciresinol.

Topics: Bacteroides; Butylene Glycols; Clostridium; Diet; Feces; Glucosides; Humans; Intestinal Mucosa; Intestines; Lignans; Species Specificity

This study investigated the effects of processing and storage on the stability of purified, flaxseed-derived secoisolariciresinol diglucoside (SDG) added to milk prior to the manufacture of different dairy products. We analyzed the effect of high-temperature pasteurization, fermentation, and milk renneting as well as storage on the stability of SDG added to milk, yogurt, and cheese. Also, the stability of SDG in whey-based drinks was studied. Added SDG was found to withstand the studied processes well. In edam cheese manufacture, most of the added SDG was retained in the whey fraction and 6% was found in the cheese curd. SDG was also relatively stable in edam cheese during ripening of 6 weeks at 9 degrees C and in yogurt during storage of 21 days at 4 degrees C. Up to 25% of added SDG was lost in whey-based drinks during storage of 6 months at 8 degrees C. We conclude that SDG can be successfully supplemented in dairy-based products.

Topics: Butylene Glycols; Dairy Products; Flax; Food Technology; Glucosides; Lignans

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

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

Previous studies have shown that feeding flaxseed (FS) or its lignan secoisolariciresinol diglucoside (SDG) to rat dams during lactation enhances the differentiation of rat mammary gland in the female offspring. This study determined whether exposure to a diet with 10% FS or SDG (equivalent to the amount in 10% FS) during suckling could protect against 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced rat mammary tumorigenesis later in life. Dams were fed the AIN-93G basal diet (BD) throughout pregnancy. After delivery, dams were randomized to continue on BD or were fed BD supplemented with 10% FS or SDG during lactation. Three-day urine of dams was analyzed for mammalian lignans. After weaning, all offspring were fed BD. At postnatal Days 49 to 51, during proestrus phase, offspring were gavaged with 5 mg of DMBA. At Week 21 post-DMBA administration, compared with the BD group, the FS and SDG groups had significantly lower (P < 0.05) tumor incidence (31.3% and 42.0% lower, respectively), total tumor load (50.8% and 62.5% lower, respectively), mean tumor size (43.9% and 67.7% lower, respectively), and tumor number (46.9% and 44.8% lower, respectively) per rat. There was a significant decreasing trend (P < 0.05) in final tumor weights in rats fed FS or SDG. The high urinary lignan excretion in dams fed with FS or SDG corresponded with the reduced tumor development. The FS and SDG groups did not differ significantly in tumor indices, indicating that the effect of FS is primarily due to its SDG. There were no significant changes in selective reproductive indices measured among dams and offspring. In conclusion, exposure to FS or SDG during suckling suppressed DMBA-induced rat mammary tumorigenesis, suggesting that exposure to lignans at this early stage of mammary gland development reduces susceptibility to mammary carcinogenesis later in life without adverse effects on selective reproductive indices in dams or offspring.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Animals, Suckling; Body Weight; Butylene Glycols; Carcinogens; Diet; Disease Models, Animal; Female; Flax; Glucosides; Lactation; Lignans; Mammary Neoplasms, Experimental; Rats; Rats, Sprague-Dawley; Reproduction

Application of stable and radioisotope precursor/tracer experiments resulted in the identification of various phenylpropanoid, monolignol, and lignan metabolites involved in the biosynthesis of the cancer chemopreventive secoisolariciresinol diglucoside (SDG; 1)-containing ester-linked "polymer(s)" in flax (Linum usitatissimum) seed. Individual analysis of size-segregated flax seed capsules at five early stages of their development provided a metabolic profile of intermediates leading to "biopolymer" biosynthesis. The use of (1)H and (13)C NMR and HRMS analyses resulted in the identification of 6a-HMG (hydroxymethyl glutaryl) SDG (17) and 6a,6a'-di-HMG SDG (18) as the two major components of the ester-linked "biopolymer(s)". Based on metabolic tracer analyses and relative radioisotopic incorporations throughout each of these five stages of seed development, a biochemical pathway is proposed from phenylalanine to SDG (1), with subsequent mono- and di-substitutions of SDG (1) with HMG CoA. These metabolites then serve as precursors for formation of the SDG-HMG ester-linked oligomers. Results from this study will facilitate future isolation and characterization of the proteins and enzymes involved in biosynthesis of the SDG-HMG ester-linked oligomers in flax seed.

Topics: Benzimidazoles; Butylene Glycols; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Flax; Gas Chromatography-Mass Spectrometry; Glucosides; Lignans; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Phenylalanine; Phenylpropionates; Plants, Medicinal; Polymers; Seeds; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stereoisomerism; Tetrazoles

Although chronic exposure to secoisolariciresinol diglycoside (SDG) was shown to alter (3)H-SDG metabolite disposition in rats, the proportion of measured radioactivity attributed to known or unknown SDG metabolites was not determined. Using HPLC and GC-MS, two experiments were conducted to determine the effect of acute (1 d) vs. chronic (10 d) SDG treatment on major urinary metabolites of (3)H-SDG in female, Sprague-Dawley rats (70-72-d-old) over a 48-h period and if new urinary metabolites were detectable in rats fed nonradioactive flaxseed or SDG. A third experiment was conducted to determine changes in postprandial blood levels of (3)H-SDG metabolites over a 24-h period with acute or chronic SDG treatment. Regardless of treatment, enterodiol, enterolactone and secoisolariciresinol accounted for 75-80% of urine radioactivity. Four potential new lignan metabolites, two of which were detected in the urine of rats fed nonradioactive flaxseed or SDG, were found. Type of treatment had no effect on levels of individual urinary metabolites of (3)H-SDG. As observed for plasma lignans in women fed flaxseed, blood radioactivity peaked at 9 h and remained high until 24 h in both treatment groups, suggesting that blood lignan kinetics might be similar with flaxseed or SDG consumption and that they were comparable between humans and rats. In conclusion, the main urinary lignan metabolites were enterodiol, enterolactone and secoisolariciresinol. Urinary composition or blood levels of radioactive lignans were not affected by the duration of SDG exposure. Thus, while chronic SDG exposure alters lignan disposition in rats, it does not change the metabolite profile.

Topics: Analysis of Variance; Animals; Butylene Glycols; Chromatography, High Pressure Liquid; Female; Gas Chromatography-Mass Spectrometry; Glucosides; Lignans; Postprandial Period; Rats; Rats, Sprague-Dawley

Flaxseed and its lignan secoisolariciresinol diglycoside (SDG) inhibit mammary tumor development in rats. Increased plasma insulin-like growth factor I (IGF-I) concentrations are associated with increased breast cancer risk. Therefore, the effect of flaxseed (5%) or SDG (1.5 mg/day) supplementation on plasma IGF-I levels was examined in rats treated with or without N-methyl-N-nitrosourea (MNU). In MNU-free rats, flaxseed and SDG reduced plasma IGF-I levels, which were inversely related to urinary lignan excretion. Only flaxseed significantly reduced plasma IGF-I concentrations in MNU-treated rats. The anticancer effect of flaxseed and SDG may be related, in part, to reductions in plasma IGF-I.

Topics: Animals; Anticarcinogenic Agents; Body Weight; Butylene Glycols; Carcinogens; Dietary Supplements; Eating; Female; Flax; Glucosides; Insulin-Like Growth Factor I; Lignans; Mammary Glands, Animal; Methylnitrosourea; Organ Size; Rats; Rats, Sprague-Dawley

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

Previous investigation demonstrated that feeding a 10% flaxseed (10F) diet during pregnancy and lactation enhanced the differentiation of highly proliferative terminal end bud (TEB) structures of rat mammary gland into less proliferative alveolar buds and lobules. From this study, it was hypothesized that the lignan component in flaxseed mediated the observed effects. Because mammary glands with more TEBs are more susceptible to carcinogens, exposure to flaxseed during early postnatal life may reduce the risk of developing mammary cancer. Our objectives were to elucidate whether exposure to flaxseed during lactation only and during pregnancy and lactation can similarly influence the differentiation of mammary gland structures and also to identify whether the lignan component of flaxseed is the biologically active agent. Offspring were exposed to a 10F diet or a dose of purified lignan equivalent to that in a 10F diet (10S) during lactation only or from lactation to postnatal Day 50. Compared with controls, exposure to 10F or 10S during lactation only or from lactation to postnatal Day 50 reduced the number of TEBs and resulted in a rise in the number of alveolar buds. In conclusion, exposure to flaxseed or its purified lignan during lactation is a critical period in which mammary gland development may be promoted by enhancing the differentiation of the mammary gland structures. However, continuous exposure, particularly to purified lignans, resulted in the most differentiation of the mammary gland. The next step is to determine whether the changes in mammary gland structures are chemopreventive in rats challenged with a carcinogen.

Topics: Analysis of Variance; Animals; Anticarcinogenic Agents; Butylene Glycols; Female; Flax; Glucosides; Lactation; Lignans; Mammary Glands, Animal; Mammary Neoplasms, Animal; Pregnancy; Random Allocation; Rats

Dietary supplementation with flaxseed or its lignan secoisolariciresinol diglycoside (SDG) has reduced dimethylbenz[a]anthracene-induced mammary tumor size and number in rats. The objective of this study was to determine whether flaxseed has a dose-dependent effect on N-methyl-N-nitrosourea (MNU)-induced mammary tumor promotion and whether this effect can be attributed to its SDG. Two days after injection with MNU (50 mg/kg body wt i.p.), female Sprague-Dawley rats were fed a high-fat (20% soybean oil) AIN-93G basal diet alone (BD) or supplemented with flaxseed (2.5% F and 5% F) or SDG by gavage [SDG in 2.5% F (LSDG) and SDG in 5% F (HSDG)] for 22 weeks. Although tumors tended to be smallest in the 5% F group throughout the experimental period, flaxseed feeding did not significantly affect tumor size, multiplicity, or incidence in comparison to BD. However, there was a dose-dependent effect of SDG on tumor multiplicity. Tumor multiplicity was lowest in the HSDG group and highest in the LSDG group throughout treatment (p < 0.05), indicating that HSDG inhibited, whereas LSDG promoted, MNU-induced mammary tumor development. Tumor invasiveness and grade were decreased in all treatment groups compared with the BD (p < 0.032). Thus, although flaxseed feeding had no significant effect on tumor growth indexes, flaxseed and SDG treatment, regardless of dose, appeared to delay the progression of MNU-induced mammary tumorigenesis. Disparities between this study and previous studies on flaxseed may be related to differences in experimental design, the use and dose of a different carcinogen, and protective effects by the alpha-linolenic acid present in the BD.

Topics: Adenocarcinoma; Animals; Butylene Glycols; Carcinogens; Diet; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Flax; Glucosides; Lignans; Mammary Neoplasms, Experimental; Methylnitrosourea; Phytotherapy; Random Allocation; Rats; Rats, Sprague-Dawley

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

Mammalian lignans from colonic bacterial action on secoisolariciresinol diglycoside (SDG) may mediate the anticarcinogenic effect of prolonged SDG feeding in rats. To elucidate lignan bioactivity, our objective was to determine 3H-SDG disposition in rats with acute or chronic SDG treatment over 48 h. After food deprivation overnight, female Sprague-Dawley rats (70-72 d old) were given a single gavage of 3H-SDG (3.7 kBq/g body weight) either immediately (acute, n = 12) or after 10 d of gavage with 1.5 mg unlabeled SDG/d (chronic, n = 12). Rats were killed at 12, 24, 36 and 48 h after gavage, and samples collected and analyzed for radioactivity by liquid scintillation counting. Radioactivity was 1- to 16-fold higher at 12 vs. 48 h for tissues, blood and gastrointestinal contents (P < 0.05). By 48 h, >80% of the recovered dose was excreted in both groups (feces > 50%, urine = 28-32%). Tissue radioactivity was highest (by 0.5- to 176-fold) in the cecum (P < 0.05). Levels in the liver, kidney and uterus (12 h) were 0.2- to 7.5-fold higher than in other nongastrointestinal tissues. At 12 h, fecal radioactivity was negligible, and cecal content, liver and adipose radioactivity were one- to threefold greater in rats with chronic SDG exposure than in those acutely exposed (P < 0.05). Blood radioactivity, present mostly in the plasma fraction (0.4% of dose), suggested that lignan concentrations could be 3000 times higher than peak estrogen levels in rats. Thus, SDG metabolites accumulated in the liver, kidney, intestinal tissues and uterus. Chronic SDG exposure delayed fecal excretion while increasing liver and adipose lignan levels.

Topics: Animals; Blood; Butylene Glycols; Feces; Female; Gastrointestinal Contents; Glucosides; Lignans; Rats; Rats, Sprague-Dawley; Time Factors; Tissue Distribution

Flaxseed, the richest source of mammalian lignan precursors, such as secoisolariciresinol diglycoside (SD), has been shown over the short term to decrease some early markers of colon cancer risk. This study determined whether over the long term flaxseed still exerts a colon cancer protective effect, whether its effect may, in part, be due to its high content of SD and whether any change in beta-glucuronidase activity plays a role in the protective effect. Six groups of male Sprague-Dawley rats were fed for 100 days either a basal high fat (20%) diet (BD), BD supplemented with 2.5 or 5% flaxseed or 2.5 or 5% defatted flaxseed (equivalent to the respective flaxseed diets) or BD with a daily gavage of 1.5 mg SD. All rats were injected with a single dose of azoxymethane (15 mg/kg body wt) 1 week prior to commencing the dietary treatments. Urinary lignan excretion, which is an indicator of mammalian lignan production, was significantly increased in the flaxseed and defatted flaxseed groups. The total activity of cecal beta-glucuronidase was significantly increased in a dose-dependent manner by the flaxseed and defatted flaxseed diet groups. Compared with the control the number of aberrant crypts per focus was significantly reduced in the distal colon of the treated rats. Four microadenomas and two polyps were observed in the control group, but not in the treated groups. The total activity of beta-glucuronidase was positively correlated with total urinary lignan excretion and negatively with the total number of aberrant crypts and the total number of aberrant crypt foci in the distal colon. There were no significant differences between the flaxseed and the corresponding defatted flaxseed groups. It is concluded that flaxseed has a colon cancer protective effect, that it is due, in part, to SD and that the protective effect of flaxseed is associated with increased beta-glucuronidase activity.

Topics: Animals; Anticarcinogenic Agents; Body Weight; Butylene Glycols; Cecum; Colonic Neoplasms; Eating; Fatty Acids; Glucosides; Glucuronidase; Hydrogen-Ion Concentration; Lignans; Male; Organ Size; Plant Extracts; Precancerous Conditions; Rats; Rats, Sprague-Dawley; Seeds

Flaxseed, a rich source of mammalian lignan precursor secoisolariciresinol-diglycoside (S.D.) and alpha-linolenic acid (ALA), has been shown to be protective at the early promotion stage of carcinogenesis. The objective of this study was to determine whether supplementation with flaxseed, its lignan or oil fractions, beginning 13 weeks after carcinogen administration, would reduce the size of established mammary tumors (present at the start of treatment) and appearance of new tumors in rats. Dietary groups consisted of the basal diet (BD, 20% corn oil) alone or supplemented with a gavage of 2200 nmol/day S.D. [S.D., equal to level in 5% flaxseed (F)], 1.82% flaxseed oil (OIL, equal to level in 5% F) or 2.5% or 5% flaxseed (2.5% F and 5% F, respectively). After 7 weeks of treatment, established tumor volume was over 50% smaller in all treatment groups (OIL, 2.5% F, 5% F, P < 0.04; S.D., P < 0.08) while there was no change in the BD group. New tumor number and volume were lowest in the S.D. (P < 0.02) and 2.5% F (P < 0.07) groups. The combined established and new tumor volumes were smaller for the S.D., 2.5% F and 5% F groups (P < 0.02) compared to the OIL and BD groups. The high negative correlation (r = -0.997, P < 0.001) between established tumor volume and urinary mammalian lignan excretion in the BD, S.D., 2.5% F and 5% F groups indicates that the reduction in tumor size is due in part to the lignans derived from the S.D. in flaxseed. However, there was no relationship between new or total tumor development and urinary lignan levels. The effect of flaxseed oil may be related to its high ALA content. In conclusion, the S.D. in flaxseed appears to be beneficial throughout the promotional phase of carcinogenesis whereas the oil component is more effective at the stage when tumors have already been established.

Topics: Animals; Antineoplastic Agents, Phytogenic; Butylene Glycols; Carcinogens; Cell Division; Female; Glucosides; Lignans; Mammary Neoplasms, Experimental; Rats; Rats, Sprague-Dawley; Seeds

A strong research effort has been devoted in recent years to the mammalian lignans enterolactone and enterodiol because of their claimed protective effects against breast and colon cancer. Some plant lignans appear to be precursors of these molecules. This paper reports a novel identification procedure for one of these precursors, the secoisolariciresinol diglucoside, in the extracts of defatted flax-seed meal. The procedure is based on high-performance liquid chromatography coupled with either ionspray or continuous-flow fast-atom bombardment mass spectrometry. The application of these techniques allowed the identification in flax seed of two isomeric forms of this precursor for the first time. The isomers are reasonable the two diastereoisomers, due to the two equivalent stereogenic centers present in the molecule.

Topics: Butylene Glycols; Chromatography, High Pressure Liquid; Glucosides; Isomerism; Lignans; Seeds; Spectrometry, Mass, Fast Atom Bombardment; Spectrophotometry, Ultraviolet

Ionspray mass spectrometry allowed the rapid detection and unambiguous identification of secoisolariciresinol diglucoside, an important mammalian lignan precursor occurring in flax seed. It was observed that this compound is embedded in a complex form, probably a complex glucoside of still undisclosed structure, among the polar solvent extractables of flax seed from which it could not be released as such without the intervention of alkaline methanolysis. Comparison of ionspray mass spectra of untreated and methanolysed flax-seed extracts confirmed methanolysis as the crucial step in setting up any further analytical or preparative procedure concerned with secoisolariciresinol diglucoside.

Topics: Butylene Glycols; Glucosides; Lignans; Mass Spectrometry; Molecular Weight; Plant Extracts; Seeds