Comparative pharmacokinetics of purified flaxseed and associated mammalian lignans in male Wistar rats.

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.

Permeability and conjugative metabolism of flaxseed lignans by Caco-2 human intestinal cells.

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 ß-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.

Flax lignans--analytical methods and how they influence our lunderstanding of biological activity.

Flaxseed (Linum usitatissimum L.) is a major source of dietary intake of lignans by virtue of the high concentrations (0.7-1.5%) that are present in the seed. The principal lignan present in flaxseed is secoisolariciresinol diglucoside (SDG), which occurs as a component of a linear ester-linked complex in which the C6-OH of the glucose of SDG is esterified to the carboxylic acid of hydroxymethylglutaric acid. Also present in flaxseed and in resulting lignan extracts are significant quantities of 2 cinnamic acid glycosides. Our emerging understanding of the biological activity of flax lignans is based on studies using a variety of materials ranging from whole ground seed to pure SDG. The underlying assumption of most of these studies is that the biological activity of flax lignans results from their conversion to the mammalian lignans enterolactone (EL) and enterodiol (ED). There are, however, several intermediate compounds generated during the digestion and metabolism of flax lignans, including SDG and its aglycones and secoisolariciresinol (Seco), that are good candidates to be the principal bioactive molecule. This review will document the history of the development of lignan analytical methods and illustrate how analytical methods have influenced the interpretation of animal and human trials and our understanding of the biological activity of flax lignans.

Pigmentation in the developing seed coat and seedling leaves of Brassica carinata is controlled at the dihydroflavonol reductase locus.

Flavonoid differences between near-isogenic lines of yellow- and brown-seeded Brassica carinata were used to identify a genetic block in seed coat and seedling leaf pigment biosynthesis. Seed coat pigment in the brown-seeded line consisted of proanthocyanidins (condensed tannins), while anthocyanin was absent. Dihydroquercetin, dihydrokaempferol, quercetin and kaempferol accumulated only in the mature seed coat of the yellow-seeded line, indicating dihydroflavonol reductase (DFR) as an element of genetic control in pigment biosynthesis. DFR transcripts from the developing seed coat in the yellow-seeded line were absent or less abundant at 5-30 days after pollination compared to transcript levels in the brown-seeded line. Seedling leaves of the yellow-seeded line exhibited reduced expression of DFR and contained less anthocyanin compared to the respective tissues from plants of the brown-seeded line when grown at 25/20 degrees C (day/night). Cooler (18/15 degrees C) growing temperatures affected seedling leaf pigmentation, mature seed coat colouration and DFR expression in the yellow-seeded line. Comparable brown-seeded line tissues were unaffected by these temperature changes. These results are suggestive of a temperature-sensitive regulator of DFR in the yellow-seeded line of Brassica carinata which ultimately affects the formation of pigments in the seedling leaves and in the mature seed coats.

Improved method for direct high-performance liquid chromatography assay of angiotensin-converting enzyme-catalyzed reactions.

A rapid and sensitive assay was developed for determination of the activity of angiotensin-converting enzyme (ACE) in the presence of inhibitory peptides present in soybean protein hydrolysates. The method utilizes reversed-phase high-performance liquid chromatography (HPLC) to separate and quantify hippuryl-histidyl-leucine (HHL) and hippuric acid (HA). HHL and HA were separated on a Symmetry C18 column by gradient elution that used mixtures of trifluoroacetic acid TFA)-acetonitrile and TFA-water as solvents. Analytical time and baseline separation of HA from HHL were improved over previous HPLC methods. In comparison to the standard spectrophotometric method, the new HPLC method obviates the need for ethyl acetate extraction of HA but requires direct injection of the ACE reaction mixture onto the HPLC column.

The effects of dietary flaxseed on the Fischer 344 rat: II. Liver gamma-glutamyltranspeptidase activity.

The effect of 10% flax chow consumption from the 30th to the 130th day after birth was examined in male Fischer 344 rats. The effects of both the high lignan/high oil Norlin strain and a high lignan/low oil Solin strain of flaxseed were compared. Physically and behaviourally there were no differences in rats belonging to the three dietary groups at any time. At 50 and 100 days of dietary exposure, blood glucose levels were the same in Norlin and Solin flax chow-fed and as well as regular chow-fed rats; there were no signs of toxicity in the Norlin and Solin flax-fed rats since their plasma levels of alanine aminotransferase were the same and equal to those of regular chow-fed rats. The activity of gamma-glutamyltranspeptidase (gammaGT) displayed an increase in the liver homogenates of flax chow-fed rats. This increase was the same in Norlin and Solin flax-fed rats at 50 and 100 days. Thus the liver effect was not oil, but lignan, likely secoisolariciresinol diglucoside (SDG), induced and was effected early on, and sustained, after flax exposure. The degree of heat activation of liver homogenate gammaGT was the same in regular chow-fed and flax chow-fed rats. Compared to liver homogenate gammaGT activity, the soluble form of gammaGT was expressed at very low levels while the plasma membrane-bound form of gammaGT was expressed at very high levels in rat liver in both regular chow-fed and flax chow-fed rats. There was no effect of flax feeding on the soluble form of liver gammaGT which was expressed at a very low level. Flax feeding effected an increase in the activity of gammaGT in isolated plasma membrane fractions which mirrored that in liver homogenates: the same degree of increase was seen in Norlin flax chow-fed and Solin flax chow-fed rats. Flax consumption effects an increase in the activity of liver gammaGT at the level of the plasma membrane which is lignan dependent, physiologically relevant and may be linked to hepatoprotection against injury through an increase in reduced glutathione.

Chemical profiling of lentil (Lens culinaris Medik.) Cultivars and isolation of compounds.

A high-performance liquid chromatography method was developed to obtain fingerprints of secondary metabolites of 12 lentil cultivars grown under the same environmental condition. Extracts (100% methanol and methanol-water (1:1)) were analyzed by RP-HPLC. Full photodiode array (191-360 nm) data were collected and used for cluster analysis. Methanol and methanol-water extracts showed slightly different clustering patterns. In the dendogram of methanol extracts, CDC Richlea appeared as an isolated group, whereas Indianhead was the isolated group in methanol-water extracts. The cultivar CDC Milestone was selected for further evaluation because of the presence of three peaks (8.9, 16.7, and 32.7 min) that were absent in other cultivars or present in very small amounts. Chromatographic separations of the methanol extract afforded several compounds including the novel 4-chloro-1H-indole-3-N-methylacetamide (13) as well as itaconic acid (3), arbutin (5), gentisic acid 5-O-[beta-d-apiofuranosyl-(1-->2)-beta-d-xylopyranoside] (9), and (6S,7Z,9R)-9-hydroxymegastigma-4,7-dien-3-one-9-O-beta-d-apiofuranosyl-(1-->2)-beta-d-glucopyranoside (14), which are described for the first time from lentils. Structures were determined by high-resolution NMR experiments.

Production of angiotensin I-converting enzyme inhibitory peptides from defatted canola meal.

To simplify the method of ACE-inhibitory peptide production, defatted canola meal was subjected to enzymatic proteolysis. Alcalase 2.4 L and protease M "Amano" were found to be the most efficient enzymes in releasing ACE-inhibitory peptides from canola proteins among 13 tested enzymes. The IC(50) values of canola protein hydrolysates ranged from 18.1 to 82.5 microg protein/mL. Differences in ACE-inhibitory activities of various protein hydrolysates reflected varied enzyme specificities. A positive correlation was determined between ACE-inhibitory activity and the degree of hydrolysis (r=0.5916, p<0.001). Ion-exchange chromatography of canola protein hydrolysate increased the protein content greater than 95% without loss of ACE-inhibitory activity. This fraction was resistant to the degradation of gastrointestinal enzyme and ACE during in vitro incubation and may be a useful ingredient in the formulation of hypotensive functional food products.

Isoflavone content and its potential contribution to the antihypertensive activity in soybean Angiotensin I converting enzyme inhibitory peptides.

A soybean angiotensin I converting enzyme (ACE) inhibitory peptide fraction was reported to have antihypertensive activity in a rat study. The purpose of the present study was to examine if the presence of isoflavones in the soybean ACE inhibitory peptide fraction may contribute to the blood-pressure-lowering property. The isoflavone concentration in soybean samples was analyzed on a C 18 reverse-phase column using a two-step gradient solvent system. Producing soybean hydrolysate led to a nearly 40% loss of isoflavones compared with the original soybean flour, but the isoflavone composition did not change and the dominant isoflavone chemicals remained as 6''-O-malonylgenistin and 6''-O-malonyldaidzin. Ion exchange chromatography affected significantly both the content and the composition of the isoflavones. The dominant isoflavones in the ion-exchanged fraction were aglycones and nonacylated isoflavones, accounting for 95.8% of the total amount of 987.7 microg/g. It was calculated that the isoflavone content in the soybean ACE inhibitory peptide fraction was 25 times less than the minimal effective isoflavone dose reported. In vitro study also showed that adding isoflavones into both soybean flour hydrolysate and soybean ACE inhibitory peptide samples to a concentration of as high as 31.5% (w/w) did not affect ACE inhibitory activity (IC 50 values). The findings along with previously published results indicated that the contribution of isoflavones in soybean ACE inhibitory peptide fraction to the blood-pressure-lowering property in a short-term feeding study might be negligible.

AAPH-mediated antioxidant reactions of secoisolariciresinol and SDG.

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.

Characterization of condensed tannins purified from legume forages: chromophore production, protein precipitation, and inhibitory effects on cellulose digestion.

To identify simple screening tools for selecting condensed tannin (CT)-containing forages as candidate sources for further study, CT were isolated from nine legumes, and their molecular weights (MW), chromophore production, capacity to precipitate bovine serum albumin (BSA) and Fraction 1 protein (Rubisco) isolated from alfalfa, and inhibition of filter paper digestion were compared. Sources were as follows: leaves of sericea lespedeza (Lespedeza cuneata Dum.-Cours.), crown vetch (Coronilla varia L.), and sainfoin (Onobrychis viciifolia Scop.); stems of hedysarum (Hedysarum alpinum L.); seeds of alfalfa (Medicago sativa L.); and whole plants of birdsfoot trefoil (Lotus corniculatus var. corniculatus L.) and three varieties of big trefoil (Lotus pedunculatus Cav.), viz., Lotus uliginosus Schkuhr, L. uliginosus var. glabriusculus, and L. uliginosus var. villosus. Molecular weights and sizes (degrees of polymerization) of the CT varied considerably within and among plant species. Average MW ranged from 3036 Da (crown vetch) to 7143 Da (lespedeza). All CT exhibited greater capacity (w/w basis) to bind alfalfa Rubisco than BSA. Relative astringencies (microg CT required to precipitate 1 mg protein) against BSA ranged from 262.5 for CT from lespedeza to 435.5 for CT from L. corniculatus, and against Rubisco, from 49.6 (sainfoin) to 108.2 (alfalfa seed). Including CT at 300 microg/ml in cultures of Fibrobacter succinogenes reduced digestion of cellulose filter paper by 19.8% (sainfoin) to 92.4% (crown vetch) and increased the specific activity of cell-associated endoglucanase. There were no correlations between inhibitory effects of CT on filter paper digestion and (1) chromophore formation during CT assay by butanol-HCl, vanillin-HCl, or H2SO4; (2) precipitation of BSA or alfalfa Rubisco; and (3) MW of CT. The most inhibitory CT for cellulose digestion included those with broad and with narrow MW distributions. Sainfoin was the most desirable source of CT, as it had the highest capacity to bind alfalfa protein and was least inhibitory to cellulose digestion by F. succinogenes. This study suggests that these properties are not easily defined via chemical means, and that biological assays using rumen bacteria may help identify those CT with properties of nutritional interest.

The effect of flax seed cultivars with differing content of alpha-linolenic acid and lignans on responses to mental stress.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSION: Flax phytoestrogens ameliorate certain responses to stress and thus may afford protection against atherosclerosis; this hypothesis should be tested in clinical trials.