Unsubstituted metallophthalocyanine catalysts for the removal of endocrine disrupting compounds using H2O2 as oxidant.

Advanced oxidation processes have become increasingly important to treat non-biodegradable compounds entering environmental waters. In recent decades, water-soluble metallophthalocyanines have been shown to catalyse H2O2-containing oxidation reactions through the production of unique reactive species, nucleophilic metal-peroxo complexes. Few reports in the literature have examined water insoluble metallophthalocyanines (MPc). The oxidative catalytic activity of water insoluble manganese- and iron-phthalocyanine (MnPc, FePc) at pH 7 has been shown through the decolourisation of methylene blue and removal of bisphenol A. These studies expand on this previous study, exploring the catalytic activity of a range of metallophthalocyanines catalysts under both acidic and neutral conditions. FePc, while only active under neutral conditions, was the best performing catalyst. This activity was significantly improved upon by the addition of acetonitrile as a co-solvent, as well as increasing the ratio of H2O2 to catalyst. MnPc was catalytically active at both pH 3 and 7. FePc and MnPc catalysts showed the ability to remove bisphenol A in the presence of dam water. Reaction rates were reduced for bisphenol A removal with FePc as a catalyst but were unchanged in the presence of MnPc. The removal of 17ß-estradiol, estrone, and coumestrol was successfully demonstrated, with greater than 96% removal of all tested EDC's achieved. This is the first reported study showing the removal of the phytoestrogen, coumestrol. Even though considerably lower concentrations of costly catalysts and oxidation reagents were used in our work, the removal extent of EDC's by the MPc-catalysed oxidation reactions achieved here compares favourably with literature.

Distribution of isoflavones and coumestrol in neglected tropical and subtropical legumes.

BACKGROUND: Isoflavones and coumestrol from dietary legumes are plant constituents showing multiple beneficial effects on humans. Owing to their ability to bind with mammalian estrogenic receptors and thereby intervention in several kinds of hormone-related cancers, they have received much attention. Soybean (Glycine max) is currently the major source of isoflavonoids in human diet. However, dozens of tropical and subtropical leguminous species remain unexplored for their isoflavonoids content. RESULTS: We have analyzed 55 extracts from 41 tropical and subtropical legume species used either in human or animal diet by high-performance liquid chromatography for the content of soy isoflavones, biochanin A, daidzein, daidzin, formononetin, genistein, genistin, sissotrin, ononin and the coumestan coumestrol. Genistein and biochanin A were the most abundant compounds. The highest content of genistein was found in aerial parts of Andira macrothyrsa, seeds of Pachyrhizus tuberosus and aerial parts of Calopogonium mucunoides (598, 250 and 184 µg g(-1), respectively) and biochanin A in aerial parts of Cratylia argentea, C. mucunoides and flowers of A. macrothyrsa (76, 53 and 40 µg g(-1), respectively). CONCLUSION: None of the samples tested was richer overall source of soy isoflavones and coumestrol than soybean; nevertheless several species (C. mucunoides or A. macrothyrsa) may serve as a promising source of individual compounds.

Development of an updated phytoestrogen database for use with the SWAN food frequency questionnaire: intakes and food sources in a community-based, multiethnic cohort study.

Phytoestrogens, heterocyclic phenols found in plants, may benefit several health outcomes. However, epidemiologic studies of the health effects of dietary phytoestrogens have yielded mixed results, in part due to challenges inherent in estimating dietary intakes. The goal of this study was to improve the estimates of dietary phytoestrogen consumption using a modified Block Food Frequency Questionnaire (FFQ), a 137-item FFQ created for the Study of Women's Health Across the Nation (SWAN) in 1994. To expand the database of sources from which phytonutrient intakes were computed, we conducted a comprehensive PubMed/Medline search covering January 1994 through September 2008. The expanded database included 4 isoflavones, coumestrol, and 4 lignans. The new database estimated isoflavone content of 105 food items (76.6%) vs. 14 (10.2%) in the 1994 version and computed coumestrol content of 52 food items (38.0%), compared to 1 (0.7%) in the original version. Newly added were lignans; values for 104 FFQ food items (75.9%) were calculated. In addition, we report here the phytonutrient intakes for each racial and language group in the SWAN sample and present major food sources from which the phytonutrients came. This enhanced ascertainment of phytoestrogens will permit improved studies of their health effects.

Quantification of five isoflavones and coumestrol in various solid agroenvironmental matrices using ¹³c3-labeled internal standards.

We developed and validated three different sample preparation and extraction methods followed by HPLC-MS/MS (negative electrospray ionization) analysis for the quantification of estrogenic isoflavones (formononetin, daidzein, equol, biochanin A, and genistein) and coumestrol in red clover, soil, and manure. Plant and manure samples were solid-liquid extracted, whereas soil was extracted with accelerated solvent extraction. Absolute recoveries were between 80 and 93%, 20 and 30%, and 14 and 91% for plant, soil, and manure samples, respectively. Relative recoveries ranged from 75 to 105% for all matrices, indicating that isotope-labeled internal standards (¹³C3-formononetin, ¹³C3-daidzein, ¹³C3-equol, ¹³C3-biochanin A, and ¹³C3-genistein) were capable to compensate for losses during analysis. The limits of detection in red clover, soil, and manure were 3-9 µg/g(dryweight(dw)), 0.6-8.2 ng/g(dw), and 34.2 ng/g(dw) to 17.0 µg/g(dw), respectively. Formononetin was the most dominant compound in red clover plants (up to 12.5 mg/g(dw)) and soil (up to 3.3 µg/g(dw)), whereas equol prevailed in manure (up to 387 µg/g(dw)).

Occurrence of phytoestrogens in municipal wastewater and surface waters.

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

Quantification of phytoestrogens in industrial waste streams.

Chemical compounds, including plant-based phytoestrogens, can function as hormone mimics and alter endocrine signaling in wildlife. In the present study, the waste streams from 19 plant-processing industries, including biofuel manufacturers, were sampled and analyzed for the phytoestrogens genistein, daidzein, coumestrol, formononetin, biochanin A, and zearalenone, via liquid chromatography/mass spectrometry. Eight of these industries contained phytoestrogens at environmentally relevant levels (≥1,000 ng/L), with the highest at approximately 250,000 ng/L. The influent and effluent streams of three municipal wastewater treatment plants receiving flow from some of these industries also were sampled and analyzed for the same phytoestrogens. It appeared that aerobic biological treatment, such as activated sludge, was able to remove these compounds from the liquid stream. Nevertheless, the effluent stream from one of the wastewater treatment plants had a phytoestrogen concentration above 1,000 ng/L. Results of the present study indicate the need for caution when designing facilities to treat the effluents from biofuel and other plant-processing industries.

Phytoestrogen content of beverages, nuts, seeds, and oils.

Phytoestrogens are secondary plant metabolites that have received increasing attention for their bioactivity, in particular due to their structural and functional similarity to 17beta-estradiol. Although urinary and plasma phytoestrogens can be used as biomarkers for dietary intake, this is often not possible in large epidemiological studies or in the assessment of general exposure in free-living individuals. Accurate information about dietary phytoestrogens is therefore important, but there are very limited data concerning food contents. In this study was analyzed a comprehensive selection of tea, coffee, alcoholic beverages, nuts, seeds, and oils for their phytoestrogen content using a newly developed sensitive method based on LC-MS incorporating (13)C 3-labeled standards. Phytoestrogens were detected in all foods analyzed, although the contents in gin and bitter (beer) were below the limit of quantification (1.5 microg/100 g). Lignans were the main type of phytoestrogens detected. Tea and coffee contained up to 20 microg/100 g phytoestrogens and beer (except bitter) contained up to 71 microg/100 g, mainly lignans. As these beverages are commonly consumed, they are a main source of dietary lignans. The results published here will contribute to databases of dietary phytoestrogen content and allow a more accurate determination of phytoestrogen exposure in free-living individuals.

Dietary phytoestrogens, including isoflavones, lignans, and coumestrol, in nonvitamin, nonmineral supplements commonly consumed by women in Canada.

Twenty-one nonvitamin, nonmineral dietary supplements commonly consumed by women in Canada were analyzed for isoflavones (formononetin, daidzein, genistein, glycitein), lignans (pinoresinol, lariciresinol, secoisolariciresinol, matairesinol), and coumestrol to complement our previously published food phytoestrogen database. Supplements containing soy or red clover had the highest concentrations of total isoflavones (728.2-35,417.0 ug/g) and total phytoestrogens (1030.1-35,517.7 ug/g) followed by licorice and licorice-containing supplements (41.3-363.3 ug/g isoflavones; 56.5-370.0 ug/g total phytoestrogens). Other supplements had considerably less isoflavones (

Estimated intakes of isoflavones and coumestrol in Korean population.

The dietary intakes and sources of isoflavones and coumestrol were estimated for each age group of Koreans based on data from the Korean Nutrition Survey conducted in 1998. For quantitative data on the levels of isoflavones and coumestrol, our previous study monitoring phytoestrogens in 220 Korean leguminous foodstuffs was employed and the median value for each food was adopted. The total isoflavones and coumestrol intake per capita was estimated as 23.3 mg/day, which constituted 14.2 mg daidzein, 6.7 mg genistein, 0.9 mg glycitein, 1.0 mg formononetin, 0.2 mg biochanin A, and 0.3 mg coumestrol. The top five foods arrowroot, soybean paste, tofu, soybean, and soybean sprout contributed to 88.2% of isoflavone intake, with the corresponding intake from each food being 8.3 mg/day, 4.9 mg/day, 2.6 mg/day, 2.5 mg/day, and 2.0 mg/day, respectively. Starting at age 3-6, the contributions of fermented soy products to the isoflavones intakes were around 30%. Soybean sprout was a major source of coumestrol intake in Koreans. Slight differences in the preference of these foods were observed among the various age groups. As regards the total isoflavone intakes, the highest value was 33.6 mg/day for people age 30-49, followed by age 50-64 (26.4 mg/day), 20-29 (21.0 mg/day), >or=65 (18.8 mg/day), 1-2 (14.5 mg/day), 7-12 (12.4 mg/day), 13-19 (10.1 mg/day), and 3-6 (8.9 mg/day). The intake levels are likely to be exceeded in groups who have preferably consumed high phytoestrogen-containing foods such as soy-protein-based infant formula and arrowroot.

Synthesis of hapten and conjugates of coumestrol and development of immunoassay.

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.

Detection of phytoestrogens in samples of second trimester human amniotic fluid.

There is widespread concern that fetal exposure to hormonally active chemicals may adversely affect development of the reproductive tract. Therefore, the present study was performed to develop the necessary analytical methods and test the hypothesis that dietary phytoestrogens can be quantified in second trimester human amniotic fluid. Amniotic fluid samples (n=59) from women (n=53) undergoing routine amniocentesis between 15 and 23 weeks of gestation were analyzed by gas chromatography/mass spectrometric (GC/MS). Analytes included the phytoestrogens daidzein, genistein, formononetin, biochanin A, and coumestrol. Dietary phytoestrogens were quantified in 96.2% of second trimester amniotic fluid samples tested. The mean (+/- standard deviation (S.D.)) concentration of daidzein and genistein in amniotic fluid was 1.44 +/- 1.34 and 1.69 +/- 1.48 ng/ml with maximum levels of 5.52 and 6.54 ng/ml, respectively. Second trimester amniotic fluid contains quantifiable levels of dietary phytoestrogens and thus is a marker of mid pregnancy fetal exposure.

Phytoestrogen content of purified, open- and closed-formula laboratory animal diets.

BACKGROUND AND PURPOSE: Phytoestrogens exert estrogenic effects on the central nervous system, induce estrus, and stimulate growth of the genital tract of female animals. Over 300 plants and plant products, including some used in laboratory animal diets, contain phytoestrogens. Therefore, the source and concentration of phytoestrogens in rodent diets were determined. METHODS: Twelve rodent diets and six major dietary ingredients were assayed for phytoestrogens (daidzein, genistein, formononetin, biochanin A, and coumestrol), using high-performance liquid chromatography. Three rodent diets recently formulated to reduce phytoestrogen content also were assayed. RESULTS: Formononetin, biochanin A, and coumestrol were not detected. Soybean meal was the major source of daidzein and genistein; their concentrations were directly correlated to the percentage of soybean meal in each diet. CONCLUSIONS: High, variable concentrations of daidzein and genistein are present in some rodent diets, and dietary phytoestrogens have the potential to alter results of studies of estrogenicity. Careful attention should be given to diet phytoestrogen content, and their concentration should be reported. A standardized, open-formula diet in which estrogenic substances have been reduced to levels that do not alter results of studies that are influenced by exogenous estrogens is recommended.

Phytoestrogen content and estrogenic effect of legume fodder.

This study is a summary of Finnish investigations of the phytoestrogen content of legume plants, red clover, white clover, alfalfa, and goat's rue. In addition to the chemical analyses, biological studies were performed. Uterine weight of immature rats was used as an indicator of the estrogenic effect of the fodder used. All red clover varieties studied contained estrogenic isoflavones, especially formononetin and biochanin-A. The phytoestrogen content varied from 1.0% to 2.5% of dry matter. The biological study of white clover showed a clear estrogenic effect not visible through chemical analysis. Alfalfa contains small quantities of formononetin and biochanin-A, but 25-65 ppm coumestrol in dry matter. The estrogenic effect of alfalfa was obvious in the biological study. Goat's rue did not contain any known phytoestrogens, and the biological study was completely negative.

Rapid HPLC analysis of dietary phytoestrogens from legumes and from human urine.

Due to growing evidence suggesting that phytoestrogens might protect against various cancers, particularly against breast and prostate cancer, it is important to measure the exposure of populations to these compounds by determining levels in food and in human tissue or body fluids to assess the possible cancer protective properties of these agents. Therefore, we developed a simple and fast procedure to extract and simultaneously hydrolyze phytoestrogens and their conjugates from food items, and present a fast and selective high-performance liquid chromatography (HPLC) method for precise determinations of the most common dietary phytoestrogens genistein, biochanin-A, daidzein, formononetin, and coumestrol using flavone as internal standard. For the first time HPLC was applied to measure these phytoestrogens and their most abundant metabolites equol and O-desmethyl-angolensin from human urine. The proposed methodology has been evaluated for losses due to thermal degradation during extraction and hydrolysis and due to sample handling during the entire work-up including solid phase extraction, and values are given for inter- and intra-assay variability. We present isoflavonoid levels of most common peas and beans used in "western" and "eastern" diets and compare isoflavonoid and coumestrol levels of raw, canned, and cooked foods which can be used in future epidemiological studies. We also determined human urinary levels with our methodology comparing values before and after soybean intake.

Liquid chromatographic determination of the plant estrogens coumestrol and isoflavones in animal feed.

A simplified procedure is described for the cleanup and determination of the 5 main plant estrogens in animal feed: coumestrol, genistein, biochanin A, daidzein, and formononetin. The estrogens are extracted with ethanol and purified on a Sep-Pak C18 cartridge. All 5 plant estrogens are separated in an isocratic liquid chromatographic system and quantitated by UV and fluorescence responses. The method is sensitive to about 2.5 ppm coumestrol and 10 ppm of the isoflavones.