Experimentally induced mastitis and metritis modulate soy bean derived isoflavone biotransformation in diary cows.

The present study compared the changes in isoflavone (daidzein and genistein) and their metabolite (equol and para-ethyl-phenol) concentrations in the blood plasma of cows with induced mastitis and metritis after feeding with soy bean. Sixteen cows were divided into four groups: control for mastitis group, cows with induced mastitis group, control for metritis group, and cows with induced metritis group. All cows were fed a single dose of 2.5 kg of soy bean and then blood samples were taken from the jugular vein for 8 h at predetermined intervals. The concentrations of soy bean-derived isoflavones and their active metabolites were measured in the blood plasma on HPLC system. ß-Glucuronidase activity in the blood plasma of cows was measured by fluorometric method. In the blood plasma of cows with induced mastitis and metritis, we found considerably higher concentrations and time-dependent increase in isoflavone metabolites (equol and para-ethyl-phenol) with reference to cyclic cows (P < 0.05). Moreover, we noticed significant decrease of genistein in the blood plasma of the cows with induced metritis compared with control cows (P < 0.05). In addition, in the blood plasma of the cows with induced metritis, we found an increase in ß-glucuronidase activity compared with control cows (P < 0.05). In conclusion, health status of the females influenced the concentrations of isoflavone metabolites in the blood plasma of the cows. Experimentally induced mastitis and metritis increased isoflavone absorption, biotransformation and metabolism. Therefore, we suggest that cows with induced mastitis and metritis are more exposed to active isoflavone metabolite actions than healthy cows.

Effect of flour extraction rate and baking on thiamine and riboflavin content and antioxidant capacity of traditional rye bread.

The effect of rye flour extraction rates and baking on thiamine and riboflavin content, and antioxidant capacity of traditional rye bread were studied and compared with white wheat flour. The content of thiamine was higher (10.9%) in rye dough formulated with dark rye flour (F-100%; extraction rate of 100%) than in rye dough formulated with brown rye flour (F-92%; extraction rate of 92%) that was similar to dough made with wheat flour. The riboflavin content in rye dough made from flour F-100% was also higher (16%) than in dough formulated with flour F-92%, and both provided larger riboflavin content than wheat dough. Baking led to reductions in thiamine of 56% for wheat bread and of 20% for both rye breads; however, this process caused only a 10% decrease in riboflavin for wheat bread and a 30% decrease for rye breads. Trolox equivalent antioxidant capacity, peroxyl radical scavenging capacity, DPPH radical scavenging activity, and Folin-Ciocalteu reducing capacity were higher in rye than in wheat dough and bread. Baking process produced slight changes in antioxidant activity, except for Superoxide Dismutase-like activity where a sharp decrease was observed. Our findings showed that rye breads are an important source of B vitamins and rye breads formulated with dark and brown flours showed better antioxidant properties than wheat bread. Therefore, rye breads should be more widely recommended in human nutrition.

Influence of fermentation conditions on glucosinolates, ascorbigen, and ascorbic acid content in white cabbage (Brassica oleracea var. capitata cv. Taler) cultivated in different seasons.

The content of glucosinolates (GLS), ascorbigen, and ascorbic acid in white cabbage (Brassica oleracea var. capitata cv. Taler) cultivated in different seasons (summer and winter) was determined, before and after spontaneous and starter-induced fermentation. Different salt concentrations (0.5% NaCl or 1.5% NaCl) were used for sauerkraut production. Glucoiberin, sinigrin, and glucobrassicin were dominating in raw white cabbage cultivated either in winter or summer seasons. Ascorbigen precursor, glucobrassicin, was found higher in cabbage cultivated in winter (2.54 micromol/g dw) than those grown in summer (1.83 micromol/g dw). Cabbage fermented for 7 d was found to contain only traces of some GLS irrespective of the fermentation conditions used. Ascorbigen synthesis occurred during white cabbage fermentation. Brining cabbage at low salt concentration (0.5% NaCl) improved ascorbigen content in sauerkraut after 7 d of fermentation at 25 degrees C. The highest ascorbigen concentration was observed in low-sodium (0.5% NaCl) sauerkraut produced from cabbage cultivated in winter submitted to either natural (109.0 micromol/100 g dw) or starter-induced fermentation (108.3 and 104.6 micromol/100 g dw in cabbages fermented by L. plantarum and L. mesenteroides, respectively). Ascorbic acid content was found higher in cabbage cultivated in summer and fermentation process led to significant reductions. Therefore, the selection of cabbages with high glucobrassicin content and the production of low-sodium sauerkrauts may provide enhanced health benefits towards prevention of chronic diseases.

Determination of quercetin and its glucosides in onion by electrochemical methods.

Quercetin (Q), quercetin-3,4'-diO-beta-glucoside (Q3,4'G), quercetin-3-O-beta-glucoside (Q3G) and quercetin-4'-O-beta-glucoside (Q4'G) were determined in onion bulbs (Allium cepa) by HPLC with amperometric detection after analysis of the hydrodynamic voltammograms of flavonoid standards within the potential range of 50-1000 mV and by cyclic voltammetry (CV) method. The hydrodynamic voltammetric profiles of flavonoids showed that the peak current of Q, Q3G, Q4'G and Q3,4'G increased rapidly when the applied potential exceeded +450 mV (vs. SCE). High sensitivity and low background current were observed at the applied potential of +950 mV (vs. SCE). The lower limits of detection (LOD) were determined at signal-to-noise ratio of 3 and showed the following values: 8.05x10(-8)M (Q), 1.08x10(-7) M (Q3G), 1.22x10(-7) M (Q4'G) and 2.6x10(-7) M (Q3,4'G). The data provided by HPLC-UV-MS confirmed the presence of Q, Q3G, Q4'G and Q3,4'G in 80% methanol extracts of lyophilised onion bulbs. The CV method was applied for the qualitative assessment of onion flavonoids followed by the determination of anodic peak potential (E(a)) of the standards. The qualitative analysis of onion flavonoids was based on the anodic peak current (I(a)) of the extracts after external standards addition. The recorded cyclic voltammograms of the above flavonoid standards showed that all four compounds had well-defined oxidation waves with peak potentials of 310, 390, 482 and 800 mV (vs. Ag/AgCl) for Q, Q3G, Q4'G and Q3,4'G in 50 mM acetate-acetic buffer (pH 5.5) in 80% methanol, respectively. The study proved applicability of the CV method for identifying Q, Q3G, Q4'G and Q3,4'G in onion.

Food content, processing, absorption and metabolism of onion flavonoids.

The question as to how far the development of chronic diseases in humans depends on diet still remains open. Simultaneously, epidemiological studies suggest the consumption of a flavonoids rich diet might decrease the risk of degenerative changes and certain diseases. The intake of this group of compounds as to quality and quantity depends on dietary habits and a widespread presence of quercetin in the diet makes this compound one of the key factors. Onion, one of the richest and most common quercetin sources, was particularly often studied in different aspects. Quercetin is present in onion mainly as glycosides, of which the distribution within the onion bulb changes in onion processing, and biological activities attracted a lot of attention. Especially antioxidative activity demonstrated in vitro was initially associated with most of the beneficial effects of quercetin on the human body. However, after ingestion quercetin undergoes extensive metabolism and microbial action resulting in its altered or degraded structure; therefore, most of the effects shown in in vitro experiments with the pure compound cannot be directly extrapolated to in vivo systems. Yet, this does not mean that quercetin simultaneously loses its positive impact on consumer health. Even after being metabolized it may still affect the redox balance by inducing antioxidative and detoxifying enzymes or compounds which may be involved in sustaining homeostasis.

Effects of phytoestrogens on testosterone secretion by Leydig cells from Bilgoraj ganders (Anser anser).

The aim of this study was to examine ganders at different stages of the reproductive season and the effect of: (1) diets with high phytoestrogen content and (2) in vitro phytoestrogen treatment on testosterone secretion by isolated Leydig cells. Thirty-six male Bilgoraj geese were fed control diets with low phytoestrogen content (containing grass meal) and diets with high phytoestrogens (containing alfalfa meal and soy). Testes were obtained from both groups of ganders at three different times of the breeding season: peak of reproductive activity (March), second half of reproductive activity (May) and beginning of photorefractoriness (July). Isolated Leydig cells were incubated with LH as well as genistein, daidzein, equol and coumestrol and the concentration of testosterone in the medium was determined by radioimmunoassay. The mean weight of testes from ganders in May and July decreased relative to their weights in March, but no significant differences among experimental groups were noted. No differences were observed in basal and LH-stimulated testosterone secretion by Leydig cells of ganders fed the control diets and the diets with higher phytoestrogen content. In July, LH did not stimulate testosterone secretion in either group. In vitro treatment with genistein, daidzein and equol (5 and 50 microM) inhibited basal and LH-stimulated testosterone production by Leydig cells from both groups. Coumestrol (5 and 50 microM) inhibited basal testosterone secretion only in March in the control group. Dietary exposure to phytoestrogens had a slight effect on in vitro testicular secretion in ganders. In vitro treatment with phytoestrogen inhibited testosterone production by Leydig cells. Genistein showed the strongest effect and coumestrol had the weakest influence on testicular secretion.

Isoflavone aglycone-rich extract without soy protein attenuates atherosclerosis development in cholesterol-fed rabbits.

The antiatherogenic effect of soy protein with intact isoflavones is well established, but the effects of isoflavones without soy protein have not been determined. We investigated the antiatherogenic effect of an isoflavone aglycone-rich extract (containing 429.4 mg/g isoflavone aglycones) without soy protein from fermented soy in cholesterol-fed rabbits. We fed 12-wk-old New Zealand white male rabbits diets containing 1 g/100 g cholesterol with 0, 0.33 or 1 g/100 g isoflavone aglycones for 8 wk. We also fed the rabbits a diet containing 1 g/100 g cholesterol with 1.09 g/100 g soy saponin-rich extract, a component other than isoflavone aglycones in the isoflavone aglycone-rich extract. Controls did not consume cholesterol, isoflavone aglycones or saponins. The isoflavone aglycone- and saponin-rich extracts did not affect the serum lipid profile of cholesterol-fed rabbits. The serum concentration of daidzein in its conjugated form was significantly higher in the high isoflavone group than in the low isoflavone group. The level of cholesteryl ester hydroperoxide (ChE-OOH) induced by CuSO(4) in plasma in the high isoflavone group was significantly less than that in the cholesterol group, and the ChE-OOH levels of LDL in the low and high isoflavone groups were significantly less than those in the cholesterol group. The ChE-OOH levels in plasma and LDL in the saponin group did not differ from the cholesterol group. In the aortic arch, the cholesterol concentration was significantly lower in the high isoflavone group, and malondialdehyde concentration was significantly lower in the low and high isoflavone groups compared with the cholesterol group; however these concentrations in the saponin group did not differ from those in the cholesterol group. The atherosclerotic lesion area of the aortic arch was significantly lower in the isoflavone groups (26.3% lower in the low isoflavone group and 36.9% lower in the high isoflavone group) than in the cholesterol group. The lesion areas were not different in the soy saponin and cholesterol groups. Immunohistochemical analysis revealed fewer oxidized LDL-positive macrophage-derived foam cells in atherosclerotic lesions in the aortic arch of isoflavone groups compared with that of the cholesterol group. These results suggest that the antioxidative action of isoflavones and their antioxidative metabolites inhibit the oxidation of LDL, thereby exerting an antiatherosclerotic effect.

Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans.

Isoflavones are contained in soybean or soy foods in two chemical forms, i.e., aglycones and glucosides. We investigated the difference in the absorption of soy isoflavone aglycones and glucosides in humans. After a single, low dose intake (0.11 mmol), the highest isoflavone concentrations in plasma were reached 2 and 4 h after ingestion of aglycones and glucosides, respectively; subjects were four men (41 y old) and four women (45 y old). The highest plasma concentration after aglycone intake was more than two times greater than that after glucoside ingestion. In a similar manner, we then compared the plasma isoflavone concentration profiles after intake of a single, high dose of isoflavones (1.7 mmol) in eight subjects (four men, 40 y old; four women, 47 y old) and found the highest plasma concentration after aglycone intake was more than five times higher than that after glucoside intake. In both high and low dose intake tests, the plasma concentration of genistein was significantly higher than that of daidzein despite the similar levels of intake. After long-term (4 wk) intakes (0.30 mmol/d), we also measured the plasma concentration of isoflavones (eight men, 45 y old). After 2 and 4 wk, these concentrations remained >100% higher after ingestion of aglycones than of glucosides. The isoflavone aglycones were absorbed faster and in greater amounts than their glucosides in humans. Isoflavone aglycone-rich products may be more effective than glucoside-rich products in preventing chronic disease such as coronary heart disease.

Soy isoflavone conjugation differs in fed and food-deprived rats.

An experiment clarifying the influence of food deprivation on the isoflavone conjugation pattern in rats was conducted. Food-deprived and fed rats were administered daidzein and genistein at 7.9 mcmol/kg body, and changes in their plasma metabolites (i.e., free compounds, sulfates, glucuronides, sulfates/glucuronides) were measured quantitatively as a function of time. In the food-deprived group, total plasma daidzein and genistein reached maximum concentrations of 20.9 +/- 4.4 and 11.4 +/- 3.1 mcmol/L, respectively, 10 min after administration, whereas in the fed group, the maxima were 2.4 +/- 0.8 mcmol/L for daidzein after 2 h and 1. 8 +/- 0.2 mcmol/L for genistein after 4 h. In both groups, there were significantly more daidzein sulfates than genistein sulfates. Moreover, depriving rats of food before daidzein and genistein administration significantly increased plasma isoflavone sulfates with simultaneous significant decreases in plasma isoflavone glucuronides compared with fed rats. Additionally, nonconjugated daidzein and genistein appeared in plasma of food-deprived rats for 1 h after administration. Plasma concentrations of conjugates having both sulfate and glucuronide moieties were not significantly different between the groups.

Factors affecting flavonoids absorption.

In experiments on rats, some of the factors affecting flavonoids absorption (solubility, glycosylation and nutritional status: fasted and not-fasted animals) were examined. Administration of quercetin with different solubilization degree showed no direct correlation between the quercetin absorption extent and solubility, i.e. despite 3 orders of difference in solubilization degree, the extent of absorption varied only about 4-fold. Absorption comparison of genistein and its glycoside genistin showed no difference in the extent of absorption; however, aglycone, in contrast to glycoside, was absorbed already from the rat stomach. Conjugation patterns (sulfation and glucuronization) of genistein metabolites demonstrated that the plasma of animals fasted prior to isoflavone administration contained significantly more sulfates and less glucuronides and mixed sulfates/glucuronides conjugates than the plasma of non-fasted animals.

Daidzein and genistein but not their glucosides are absorbed from the rat stomach.

Absorption of isoflavone aglycones and glucosides was compared in rats. Daidzein, genistein, daidzin and genistin were orally administered at a dose of 7.9 micromol/kg in 25 mM Na2CO3 and next their metabolite concentration in blood plasma was monitored for 30 min. After isoflavone glucosides administration, their metabolites appeared in plasma with a few minutes delay as compared to aglycones, which suggested that aglycones, but not glucosides, were absorbed already in the rat stomach. This observation was confirmed when absorption site was restricted solely to the stomach and absorption was shown to be independent of the vehicle pH used for administration.

Quercetin metabolites inhibit copper ion-induced lipid peroxidation in rat plasma.

The oxidative susceptibility of plasma obtained from rats after intragastric administration of quercetin was studied to know whether or not quercetin acts as an in vivo antioxidant after metabolic conversion. Quercetin was raised in the rat blood plasma essentially as glucuronide and/or sulfate conjugates. The plasma obtained from rats after quercetin administration was more resistant against copper sulfate-induced lipid peroxidation than the control plasma on the basis of the accumulation of cholesteryl ester hydroperoxides and the consumption of alpha-tocopherol. The results strongly suggest that some conjugated metabolites of quercetin act as effective antioxidants when plasma is subject to metal ion-induced lipid peroxidation.

Accumulation of (-)-epicatechin metabolites in rat plasma after oral administration and distribution of conjugation enzymes in rat tissues.

Absorption of orally administered (-)-epicatechin (EC) in rats was studied to obtain plasma pharmacokinetic profiles of EC metabolites. Rats were administered 172 micromol/kg body weight of EC, and blood was collected from the tail for 8 h after administration. Seven groups of compounds possessing the basic structure of EC were identified by using a combination of enzymatic hydrolysis, HPLC and electron impact mass spectrometry. Metabolites were quantified with a new, simple and sensitive method using HPLC with electrochemical detection. Ingested EC was absorbed from the alimentary tract and was present in the rat common blood circulation in the form of glucuronide and/or sulfate conjugates. The activity of conjugative enzymes in rat tissues was studied. The highest activity of glucuronosyltransferase was found in the intestinal mucosa of both of the small and large intestine; the highest activity of phenolsulfotransferase occurred in the liver, and that of catechol-O-methyl transferase was found in the liver and kidney. It has been proposed that the first detoxification step of dietary EC, namely, glucuronidation, occurs at the level of the intestinal mucosa in rats, and EC enters the common blood circulation exclusively in the glucuronized form. The compound is then sulfated in the liver and methylated in the liver and kidney. Because ingested EC undergoes extensive conjugation, its biological activities previously demonstrated in vitro may not be occurring in in vivo systems.