[Show abstract][Hide abstract] ABSTRACT: The Arabidopsis transparent testa (tt) mutant tt19-4 shows reduced seed coat colour, but stains darkly with DMACA and accumulates anthocyanins in aerial tissues. Positional cloning showed that tt19-4 was allelic to tt19-1 and has a G-to-T mutation in a conserved 3'-domain in the TT19-4 gene. Soluble and unextractable seed proanthocyanidins and hydrolysis of unextractable proanthocyanidin differ between wild-type Col-4 and both mutants. However, seed quercetins, unextractable proanthocyanidin hydrolysis, and seedling anthocyanin content, and flavonoid gene expression differ between tt19-1 and tt19-4. Transformation of tt19-1 with a TT19-4 cDNA results in vegetative anthocyanins, whereas TT19-4 cDNA cannot complement the proanthocyanidin and pale seed coat phenotype of tt19-1. Both recombinant TT19 and TT19-4 enzymes are functional GSTs and are localized in the cytosol, but TT19 did not function with wide range of flavonoids and natural products to produce conjugation products. We suggest that the dark seed coat of Arabidopsis is related to soluble proanthocyanidin content and that quercetin holds the key to the function of TT19. In addition, TT19 appears to have a 5' GSH-binding domain influencing both anthocyanin and proanthocyanidin accumulation and a 3' domain affecting proanthocyanidin accumulation by a single amino acid substitution.
[Show abstract][Hide abstract] ABSTRACT: Structures for nine compounds were elucidated in seed coats of two genetically related Brassica carinata lines. The yellow-seeded line accumulated monomeric kaempferols, phenylpropanoids, and lignans, while extractable and unextractable proanthocyanidins and a high-performance liquid chromatography peak containing polymeric-like quercetin/lignan structures were strongly reduced. The brown-seeded line accumulated large amounts of both types of proanthocyanidins (extractable and unextractable), as well as phenylpropanoids and lignans equivalent to the amounts in the yellow-seeded seed coats, but the brown-seeded seed coats lacked kaempferols. A Brassica napus 15K oligoarray experiment indicated that yellow-seeded siliques had more extreme gene expression changes and a 2.4-fold higher number of upregulated genes than brown-seeded siliques, including a host of transcription factors and genes with unknown function. Transcripts for six flavonoid genes (CHS, F3H, FOMT, DFR, GST, and TTG1) were lower and two (F3'H and FLS) were higher in yellow-seeded siliques, but expression of CHI, PAP1, and phenylpropanoid genes was unchanged.
Journal of Agricultural and Food Chemistry 10/2010; 58(20). DOI:10.1021/jf102208a · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Exposure of B. carinata seedlings to increasing concentrations of a non-physiological ion, lithium, showed significant effects on the germination rate, root length, chlorophyll content and fresh weight in brown-seeded and yellow-seeded near-isogenic lines. Metal content analysis and phytochemical profiling indicated that lithium hyper-accumulated and the lipid and phenolic composition dramatically changed in brown-seeded seedlings. Here, sinapic acid esters and chloroplast lipid were replaced by benzoate derivatives, resveratrol and oxylipins after lithium exposure. In contrast, the yellow-seeded plants maintained the same phenolic and lipid composition before and after exposure to lithium and did not tolerate the high metal concentrations tolerated by the brown-seeded line. Microarray analysis using a Brassica napus 15,000 expressed sequence tag (EST) array indicated a total of 89 genes in the brown-seeded line and 95 genes in the yellow-seeded line were differentially expressed more than 20-fold after treatment of B. carinata seedlings with lithium chloride and more than 1083 genes with expression changes greater than 2-fold. The putative functions of the differentially expressed genes included proteins involved in defense, primary metabolism, transcription, transportation, secondary metabolism, cytochrome P450, as well as proteins with unknown functions. Transcriptome changes between yellow-seeded and brown-seeded B. carinata seedlings after lithium chloride exposure indicated that the two lines responded differently to lithium treatment. The expression patterns generally supported the phytochemical data. From the results of this study, B. carinata brown-seeded germplasm showed an ability to survive under moderately high concentrations of lithium chloride (>150 mM). The ability to accumulate this metal ion suggests brown-seeded B. carinata has some potential in phytoremediation of lithium-contaminated water and soil.
[Show abstract][Hide abstract] ABSTRACT: Improving our knowledge of plant metal metabolism is facilitated by the use of analytical techniques to map the distribution of elements in tissues. One such technique is X-ray fluorescence (XRF), which has been used previously to map metal distribution in both two and three dimensions. One of the difficulties of mapping metal distribution in two dimensions is that it can be difficult to normalize for tissue thickness. When mapping metal distribution in three dimensions, the time required to collect the data can become a major constraint. In this article a compromise is suggested between two- and three-dimensional mapping using multi-angle XRF imaging.
A synchrotron-based XRF microprobe was used to map the distribution of K, Ca, Mn, Fe, Ni, Cu and Zn in whole Arabidopsis thaliana seeds. Relative concentrations of each element were determined by measuring fluorescence emitted from a 10 microm excitation beam at 13 keV. XRF spectra were collected from an array of points with 25 or 30 microm steps. Maps were recorded at 0 and 90 degrees , or at 0, 60 and 120 degrees for each seed. Using these data, circular or ellipsoidal cross-sections were modelled, and from these an apparent pathlength for the excitation beam was calculated to normalize the data. Elemental distribution was mapped in seeds from ecotype Columbia-4 plants, as well as the metal accumulation mutants manganese accumulator 1 (man1) and nicotianamine synthetase (nasx).
Multi-angle XRF imaging will be useful for mapping elemental distribution in plant tissues. It offers a compromise between two- and three-dimensional XRF mapping, as far as collection times, image resolution and ease of visualization. It is also complementary to other metal-mapping techniques. Mn, Fe and Cu had tissue-specific accumulation patterns. Metal accumulation patterns were different between seeds of the Col-4, man1 and nasx genotypes.
Annals of Botany 01/2008; 100(6):1357-65. DOI:10.1093/aob/mcm205 · 3.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The flaxseed lignans secoisolariciresinol (SECO) and its diglucoside secoisolariciresinol diglucoside (SDG) are reported to
have a number of health benefits associated with their consumption that have in part been attributed to their antioxidant
properties. In this study the relative antioxidant capacity of the flaxseed lignans vs. BHT was determined in two model systems.
First, an antioxidants stoichiometric value was determined for SECO and SDG in a liposomal system as a mimic of lipid peroxidation.
Stoichiometric values for SECO (1.5) and SDG (1.2) vs. BHT (2.0) were measured from the lag time for the formation of conjugated
dienes; all values were significantly different (P<0.01). Second, the ability of flaxseed lignans to prevent oxidative degradation of canola oil was determined. Samples were
stored at room temperature and analyzed at 30-d intervals over 120 d using a Rancimat™ analyzer. The lignans prevented degradation of canola oil, as measured by induction time, in a concentration-dependent manner.
Although SECO demonstrated a trend for better protection than either SDG or polymer containing SDG, they were not significantly
different (P>0.01). There was also no significant difference between SECO or SDG and BHT, suggesting flaxseed lignans may be good alternatives
to minimize rancidity in oil-based food products.
[Show abstract][Hide abstract] ABSTRACT: The identification of genes involved in metal metabolism in plants requires the 'screening' of thousands of genetic variants. While inductively coupled plasma mass-spectroscopy has been used to identify variants with an altered total metal concentration, a more convenient high-throughput technique capable of examining individual seeds (or other tissues) would be useful. Here, the high brightness of synchrotron radiation has been utilised to examine relative metal concentrations in seeds of the genetically well characterised plant Arabidopsis thaliana. The relative concentrations of Mn, Fe, Ni, Cu and Zn in individual seeds were determined using a 500 microm x 500 microm beam. Metal concentrations were normally distributed, except where metal-containing dust contaminated the samples. Neither seed orientation nor genetic background (from three 'wild type' variants with different genetic lineages) had a significant affect on the Zn-normalised metal concentration. No advantages, such as the observation of tissue-specific metal accumulation, were obtained by using a 50 microm x 50 microm beam. A high-throughput proof-of-concept experiment was demonstrated that could be used to screen libraries of genetic variants for individuals with altered metal concentrations. Further work is required to standardise the technique before screening of libraries is possible.
[Show abstract][Hide abstract] ABSTRACT: A new method has been developed for the extraction of volatiles from plant materials and tested on seedling tissue and mature leaves of Arabidopsis thaliana, pine needles and commercial mixtures of plant volatiles. Volatiles were extracted with n-pentane and then subjected to quick distillation at a moderate temperature. Under these conditions, compounds such as pigments, waxes and non-volatile compounds remained undistilled, while short-chain volatile compounds were distilled into a receiving flask using a high-efficiency condenser. Removal of the n-pentane and concentration of the volatiles in the receiving flask was carried out using a Vigreux column condenser prior to GC-MS. The method is ideal for the rapid extraction of low-boiling-point volatiles from small amounts of plant material, such as is required when conducting metabolic profiling or defining biological properties of volatile components from large numbers of mutant lines.
[Show abstract][Hide abstract] ABSTRACT: Dietary modification contributes significantly in reducing cardiovascular disease (CVD) risk factors including lowering cholesterol and atherosclerosis. The purpose of this study was to investigate the effects of flaxseed, a rich source of lignans, alpha-linolenic acid and soluble fiber mucilage, on the prevention of ovariectomy-induced rise in total cholesterol and atherosclerotic lesions.
Seventy-two 6-month-old female Golden Syrian hamsters were either ovariectomized (ovx) or sham-operated (sham) and randomly assigned to six groups (n = 12): sham, ovx, or ovx plus either 17beta-estradiol (E(2), 10 microg/kg body weight) or semi-purified diet adjusted for macronutrients and fiber to contain one of the three doses of flaxseed (7.5, 15, or 22.5%) for 120 days.
Ovariectomy significantly elevated plasma total-, HDL-, and free-cholesterol concentrations. Similar to estrogen, all doses of flaxseed were effective in preventing the ovx-induced rise in plasma total cholesterol. Triglyceride concentrations were significantly higher in the flax-fed hamsters. There were no significant differences in plasma non-HDL- and esterified-cholesterol among the treatment groups. Ovariectomy also increased the number of hamsters with lesions and the aortic fatty streak area. All three doses of flaxseed reduced the fatty streak area and the incidence of lesions to levels similar to the sham group.
The findings of this study show that flaxseed is beneficial in reducing plasma cholesterol and plaque formation induced by ovarian hormone deficiency.
[Show abstract][Hide abstract] ABSTRACT: Phytoestrogens offer a possible alternative to hormone replacement therapy. Flax seed contains large quantities of a phytoestrogen precursor, secoisolariciresinol diglucoside (SDG), as well as large quantities of alpha-linolenic acid; these factors may be protective against vascular disease. We have previously shown that the rise in blood pressure during mental stress is a strong predictor of atherosclerosis progression.
35 postmenopausal women with vascular disease, 62 +/- 8 years of age, were treated in a random-sequence double-blind Latin square crossover study comparing three strains of flax seed: Flanders (low in lignan and high in alpha-linolenic acid), Linola 989 (high in lignan and low in alpha-linolenic acid) and AC Linora (intermediate in both lignan and alpha-linolenic acid).
Compared to the pre-treatment baseline diet, all three strains of flax significantly reduced blood pressure during mental stress induced by a frustrating cognitive task (Stroop color-word interference task) (p = 0.004). Linola 989, the strain highest in lignan and lowest in alpha-linolenic acid, was associated with the least increase in peripheral resistance during stress, the greatest reduction in plasma cortisol during stress and the smallest increase in plasma fibrinogen during mental stress.
Flax phytoestrogens ameliorate certain responses to stress and thus may afford protection against atherosclerosis; this hypothesis should be tested in clinical trials.
Journal of the American College of Nutrition 01/2004; 22(6):494-501. DOI:10.1080/07315724.2003.10719327 · 1.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The flavonoid profiles of 22 accessions of Camelina sativa and five other crucifer species, Crambe abyssinica, Crambe hispanica, Thlaspi arvense, Brassica napus, and Sinapis alba, were studied by a combination of liquid, paper and thin layer chromatography. Flavonoids were confirmed by comparison of their characteristics, including colour under UV light, changes to colour under UV with fuming in NH3 vapour, UV spectra and comparison of RF-values, with those of authentic standards. HPLC-mass spectroscopic data were obtained to confirm identities of several compounds. Flavonoids present in several other crucifer species were identified by TLC only. We report the accumulation of derivatives of the flavonols, quercetin, in C. sativa; quercetin and kaempferol in C. hispanica var. glabrata; quercetin, kaempferol and isorhamnetin in B. napus; and kaempferol and isorhamnetin in S. alba. Derivatives of the flavones, apigenin and luteolin, accumulate in C. abyssinica, C. hispanica var. hispanica and T. arvense leaves.
[Show abstract][Hide abstract] ABSTRACT: Lignans have been part of both diet and herbal medicines for centuries. It is only in the last half century that phytochemists have described the structures of the lignans. Pharmacologists have only become interested in the biological activity of lignans in the last few decades. Much of the early interest focused on podophyllotoxin type lignans and their derivatives. Recent literature has recorded very many new lignans or lignan derivatives with a diverse range of biological activities. In 1955, the isolation from flax seed and the structure of the lignan derivative secoisolariciresinol diglucoside (SDG) was reported. Possible biological activity of SDG, and the mammalian lignan metabolites, enterolactone and enterodiol, was initially reported about 20 years later. During the next 30 years, there has been extensive research on the biological effects of both flax seed and rye lignans since both are metabolized into the mammalian lignans. Research on the activity of lignans on breast, colon, prostate and thyroid cancer has generally shown beneficial effects although there are some studies with either no conclusive or negative effect. Lignans have been shown to have positive effects in lowering relative risk factors for heart disease. Use of flax seed or SDG has been shown to have positive effects in both lupus and polycystic kidney disease models. Studies of both type I and II diabetes models have reported positive results when using SDG. Flax seed has also been reported to be hepatoprotective. Reproductive effects have been observed with flax seed or SDG and have been found to be dose and time related. There are many possible mechanistic explanations for the observed bioactivities including involvement in hormonal metabolism or availability, angiogenesis, anti-oxidation and gene suppression.
[Show abstract][Hide abstract] ABSTRACT: Three anthocyanin regulatory genes of maize (Zea mays; Lc, B-Peru, and C1) were introduced into alfalfa (Medicago sativa) in a strategy designed to stimulate the flavonoid pathway and alter the composition of flavonoids produced in forage. Lc constructs included a full-length gene and a gene with a shortened 5'-untranslated region. Lc RNA was strongly expressed in Lc transgenic alfalfa foliage, but accumulation of red-purple anthocyanin was observed only under conditions of high light intensity or low temperature. These stress conditions induced chalcone synthase and flavanone 3-hydroxylase expression in Lc transgenic alfalfa foliage compared with non-transformed plants. Genotypes containing the Lc transgene construct with a full-length 5'-untranslated region responded more quickly to stress conditions and with a more extreme phenotype. High-performance liquid chromatography analysis of field-grown tissue indicated that flavone content was reduced in forage of the Lc transgenic plants. Leucocyanidin reductase, the enzyme that controls entry of metabolites into the proanthocyanidin pathway, was activated both in foliage and in developing seeds of the Lc transgenic alfalfa genotypes. Proanthocyanidin polymer was accumulated in the forage, but (+)-catechin monomers were not detected. B-Peru transgenic and C1 transgenic populations displayed no visible phenotypic changes, although these transgenes were expressed at detectable levels. These results support the emerging picture of Lc transgene-specific patterns of expression in different recipient species. These results demonstrate that proanthocyanidin biosynthesis can be stimulated in alfalfa forage using an myc-like transgene, and they pave the way for the development of high quality, bloat-safe cultivars with ruminal protein bypass.
[Show abstract][Hide abstract] ABSTRACT: Samples of commercially prepared white, whole wheat, flax, and multigrain breads were analyzed by a rapid RP-HPLC method for the presence of the lignan secoisolariciresinol diglucoside (SDG). SDG was detected only in products containing flax, with concentrations ranging from 0.06 to 1.98 microM/g of DW (19-602 microM/loaf). Full-fat flax meal, powdered aqueous alcohol extracts of flax seed, and SDG were added to a white bread mix and baked into loaves in a domestic bread maker. Quantitative recovery of SDG from the test breads was observed when SDG was added; however, when flax meal or aqueous alcohol extracts were added, only 73-75% of the theoretical yield of SDG was recovered. SDG was also detected in commercially prepared flax cookies, bagels, and muffins with concentrations ranging from 0.26 to 2.93 microM/g of DW. The extent of grinding of the flax seed was also shown to have a significant effect on the recovery of SDG from both flax meal breads and baked goods, with extraction of SDG from finely ground samples greater than that from course material.
Journal of Agricultural and Food Chemistry 10/2000; 48(9):4048-52. DOI:10.1021/jf990922p · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen species (ROS) have been implicated in the development of streptozotocin (STZ)-induced diabetes mellitus. Secoisolariciresinol diglucoside (SDG) isolated from flaxseed is an antioxidant. An investigation was made of the effects of SDG on the development of STZ-induced diabetes in rat, to determine if SDG can prevent/reduce the development of diabetes and if this prevention/reduction is associated with reduction in oxidative stress.
The rats were divided into 4 groups: Group I, Control; Group II, SDG (22 mg/kg body wt, orally) for 24 days; Group III, STZ (80 mg/kg intraperitoneally); Group IV, SDG in the dose similar to Group II three days prior to STZ and 21 days thereafter. Oxidative stress was assessed by measuring serum and pancreatic lipid peroxidation product malondialdehyde (MDA), pancreatic antioxidant reserve (pancreatic-CL) and oxygen free radical producing activity of white blood cells (WBC-CL). A diagnosis of diabetes was made on the basis of glucosuria and was confirmed at the time of sacrifice (21 days after STZ treatment) by the presence of hyperglycemia. At the end of the protocol blood samples were collected for estimation of glucose, MDA and WBC-CL, and pancreas were removed for estimation of MDA and antioxidant reserve.
Incidence of diabetes was 100% in Group III and 25% in Group IV. SDG prevented the development of diabetes by 75%. Development of diabetes was associated with an increase in serum and pancreatic MDA, and in WBC-CL, and a decrease in pancreatic antioxidant reserve. Prevention of diabetes by SDG was associated with a decrease in serum and pancreatic MDA and WBC-CL and an increase in pancreatic antioxidant reserve.
These results suggest that STZ-induced diabetes is mediated through oxidative stress and that SDG is effective in reducing the STZ-induced diabetes mellitus.
[Show abstract][Hide abstract] ABSTRACT: Flaxseed has renoprotective effects in animal and human lupus nephritis. We have recently extracted the lignan precursor (secoisolariresinol diglucoside) (SDG) to determine if this more palatable derivative of flaxseed would exert renoprotection similar to the whole flaxseed in the aggressive MRL/lpr lupus mouse model.
131 MRL/lpr mice were randomly assigned to saline gavage, 600, 1,200 and 4,800 microg lignan gavage groups. At 7 weeks, 6 animals underwent platelet aggregating factor (PAF) lethal challenge and 40 were studied with urine collection to determine the levels of secoisolariresinol, enterodiol and enterolactone in the gavaged animals. A baseline study of 10 saline gavaged animals took place at 6 weeks. 25 animals in the saline gavage, 600 and 1200 microg lignan groups were studied at 14 and 22 weeks for GFR, spleen lymphocyte S-phase and organ weight studies.
Metabolic studies indicated that secoisolariresinol is the major metabolite absorbed and the lowest lignan dose provides a lengthening in survival for the PAF lethal challenge. Body weight, fluid and water intake studies demonstrated that the lignan was well tolerated. Changes in proteinuria, GFR and renal size showed a time- and dose-dependent protection for the lignan precursor. Cervical lymph node size and spleen lymphocyte cells in the S-phase demonstrated modest dose-dependent reductions in the lignan gavaged groups.
SDG was converted in the gut to secoisolariresinol, which was absorbed and well tolerated by the MRL/lpr mice. Renoprotection was evidenced, in a dose-dependent fashion, by a significant delay in the onset of proteinuria with preservation in GFR and renal size. This study suggests that SDG may have a therapeutic role in lupus nephritis.
[Show abstract][Hide abstract] ABSTRACT: Flaxseed (Type I flaxseed) with 51-55% alpha-linolenic acid in its oil and richest source of plant lignans, has been shown to reduce hypercholesterolemic atherosclerosis by 46% without lowering serum lipids. Antiatherogenic activity was claimed to be due to its alpha-linolenic acid and/or lignan content. If alpha-linolenic acid component of flaxseed is responsible for antiatherogenic activity, then, CDC-flaxseed (Type II flaxseed) which has similar oil and lignan content but has very little (2-3% of the total oil) alpha-linolenic acid would have no antiatherogenic effect. An investigation, therefore, was made of Type II flaxseed on high cholesterol diet-induced atherosclerosis and serum lipids [total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), very low-density lipoprotein cholesterol (VLDL-C)] in rabbits. Rabbits were assigned to four groups: Group I, Control; Group II, Type II flaxseed diet (7.5 g/kg orally daily); Group III, 1% cholesterol diet; Group IV, 1% cholesterol diet supplemented with Type II flaxseed (7.5 g/kg orally daily). Blood samples were collected before (0 time) and after 4 and 8 weeks of experimental diets for measurement of serum lipids. Aorta was removed at the end of 8 weeks for assessment of atherosclerotic plaques. Serum TC, LDL-C, TC/HDL-C, and LDL-C/HDL-C were lower in Group IV as compared to Group III by 14 and 31%, 17 and 32%, 28 and 34% and 24 and 32%, respectively, at 4 and 8 weeks. HDL-C was not affected by Type II flaxseed in hypercholesterolemic rabbit. TG and VLDL-C were markedly increased in Group IV as compared to Group III. Type II flaxseed reduced the development of atherosclerosis by 69%. Histological changes in the atherosclerotic regions were qualitatively similar in Groups III and IV. Results indicate that reduction in hypercholesterolemic atherosclerosis by Type II flaxseed is due to a decrease in serum TC and LDL-C. In conclusion, antiatherogenic activity of Type II flaxseed is not due to alpha-linolenic acid.