Effective methods for increasing coumestrol in soybean sprouts.

Coumestrol (CM), a biologically active compound found in Leguminosae plants, provides various human health benefits. To identify easy and effective methods to increase CM content in vegetables, we developed a quantitative analysis method using high-performance liquid chromatography (HPLC). Using this method, we found that soybean sprouts (1.76 ± 0.13 µg/g) have high CM contents among nine vegetables and evaluated the difference in CM contents between two organs of the sprouts: cotyledons and hypocotyls. Next, soybean sprouts were cultivated under different light, temperature, and water conditions and their CM contents were evaluated. CM content was higher in hypocotyls (4.11 ± 0.04 µg/g) than in cotyledons. Cultivating soybean sprouts at 24°C enhanced CM content regardless of light conditions, the growth of fungi and bacteria, and sprout color. Thus, we identified methods of soybean sprout cultivation to increase CM content, which may provide health benefits and enhance value.

Transcriptomic changes in green bean pods against grey mould and white rot diseases via field application of chemical elicitor nanoparticles.

The authors tested the efficacy of two salt nanoparticles (NPs), namely, copper dioxide (CuO) and tri-calcium phosphate [Ca3(PO4)2] to induce resistance in green bean pods against grey mould and white rot diseases caused by Botrytis cinerea and Sclerotinia sclerotiorum, respectively. High amounts of phytoalexins, kievitone, coumestrol, phaseollidin, 6-ά-hydroxyphaseollin, and phaseollin, were detected in naturally infected and artificially inoculated green bean pods in response to the tested NPs. Green bean plants treated in the field with CuO and Ca3(PO4)2 NPs had the highest mRNA quantity of all the studied defence genes, receptor-like kinase (PvRK20), pathogenesis-related protein (PR1), 1,3-ß-D-glucanase (pvgluc), polygalacturonase inhibitor protein (PvGIP), and alpha-dioxygenase (a-DOX) than that of the control group. CuO NPs followed by Ca3(PO4)2 NPs at 0.15 mg ml-1 were the most potent in increasing the transcriptomic levels of pk20, DOX, PR1, PvGIP, and pvgluc. Field applications of both chemical elicitor NPs exhibited a non-genotoxic effect on the Paulista green bean DNA using eight ISSR primers. The field application of the studied NPs could effectively extend the shelf life of green bean pods by up to 21 days at 7 ± 1°C during marketing and export due to its potent effect against grey mould and white rot diseases.

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.

A bioanalytical HPLC method for coumestrol quantification in skin permeation tests followed by UPLC-QTOF/HDMS stability-indicating method for identification of degradation products.

Coumestrol is present in several species of the Fabaceae family widely distributed in plants. The estrogenic and antioxidant activities of this molecule show its potential as skin anti-aging agent. These characteristics reveal the interest in developing analytical methodology for permeation studies, as well as to know the stability of coumestrol identifying the major degradation products. Thus, the present study was designed, first, to develop and validate a versatile liquid chromatography (HPLC) method to quantify coumestrol in a hydrogel formulation in different porcine skin layers (stratum corneum, epidermis, and dermis) in permeation tests. In the stability-indicating test coumestrol samples were exposed to stress conditions: temperature, UVC light, oxidative, acid and alkaline media. The degradation products, as well as the constituents extracted from the hydrogel, adhesive tape or skin were not eluted in the retention time of the coumestrol. Hence, the HPLC method showed to be versatile, specific, accurate, precise and robust showing excellent performance for quantifying coumestrol in complex matrices involving skin permeation studies. Coumestrol recovery from porcine ear skin was found to be in the range of 97.07-107.28 µg/mL; the intra-day precision (repeatability) and intermediate precision (inter-day precision), respectively lower than 4.71% and 2.09%. The analysis using ultra-performance liquid chromatography coupled to a quadrupole time-of-flight high definition mass spectrometry detector (UPLC-QTOF/HDMS) suggest the MS fragmentation patterns and the chemical structure of the main degradation products. These results represent new and relevant findings for the development of coumestrol pharmaceutical and cosmetic products.

[UPLC-MS / MS for the pharmacokinetics of icariin in rats].

OBJECTIVE: To establish an ultra-performance liquid chromatographytandem mass spectrometry( UPLC-MS / MS) method for the quantification of icariin( ICA), and investigate pharmacokinetics of ICA in rats following multiple oral administration. METHODS: ICA and an internal standard coumestrol( CMT) were extracted from rat plasma using ethyl acetate and separated on a BEH C18( 50 mm × 2. 1mm, 1. 7 µm) column using a gradient mobile phase of acetonitrile containing 0. 1%( V / V)formic acid and water containing 2 mmol / L ammonium formate at a flow rate of 0. 3m L / min. In negative electrospray ionization mode, multiple reaction monitoring of the precursor-product ion transitions of m / z 675. 6→351. 1 for ICA, 267. 0 →211. 1 for CMT was used for the quantification. Plasma was collected after rats were orally administered with ICA at multiple doses of 50 mg / kg. RESULTS: The linear calibration curve was achieved in a concentration range of 0. 5-50 ng / m L with a lower limit of quantification of0. 5 ng / m L. The value of intra- and inter-day precision was less than 11. 3% and accuracy fell in the ranges of 94. 3%-98. 7%. The recovery ranged from 81. 3% to85. 2% and the matrix effects from 94. 3% to 103. 2%. After oral administration of ICA to rats, t1 /2was( 1. 68 ± 0. 29) h, Cmaxwas( 29. 6 ± 5. 3) ng / m L, tmaxwas( 1. 00 ± 0. 00) h, AUC0- twas( 88. 4 ± 13. 9)( h·ng) / m L. CONCLUSION: The method is specific and accurate, suitable for preclinical pharmacokinetics of ICA.

Occurrence of isoflavonoids in Brazilian common bean germplasm (Phaseolus vulgaris L.).

Common bean (Phaseolus vulgaris) is present in the daily diet of various countries and, as for other legumes, has been investigated for its nutraceutical potential. Thus, 16 genotypes from different gene pools, representing seven types of seed coats and different responses to pathogens and pests, were selected to verify their isoflavone contents. The isoflavonoids daidzein and genistein and the flavonols kaempferol, myricetin, and quercetin were found. Grains of the black type showed the highest concentrations of isoflavonoids and were the only ones to exhibit daidzein. IAC Formoso, with high protein content and source of resistance to anthracnose, showed the greatest concentration of genistein, representing around 11% of the content present in soybean, as well as high levels of kaempferol. Arc 1, Raz 55, and IAC Una genotypes showed high content of coumestrol. The results suggest the use of IAC Formoso to increase the nutraceutical characteristics in common bean.

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.

[Simultaneous determination of four kinds of furocoumarins in cosmetics by high performance liquid chromatography].

OBJECTIVE: A high performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of 4 furocoumarins, including 8-methoxycoumarin, 5-methoxycoumarin, trioxsalen and imperatorin in cosmetics. METHODS: The separation was carried on Diamonsil C18 column (4.6 mm x 250 mm, 5 microm) by gradient elution with methanol-water as mobile phase at a flow rate of 1.0 ml/min and column temperature of 30 degrees C. The analysis of 4 furocoumarins was performed by HPLC with diodearray detector at the wavelength of 248 nm. RESULTS: Identification of 4 furocoumarins was achieved by retention time, and quantification analysis was performed by the external standard method. The limits of quantitation (LOQ) for 4 furocoumarins were 0.54, 0.52, 0.58 and 0.50 microg/g, respectively. The method showed a good linearity in the range of 0.05-10 microg/ml with correlation coefficients more than 0.999. The mean recoveries at spiked levels were in the range of 89.9%-105.2% with RSDs of 0.20%-1.08%. CONCLUSION: The method was accurate and simple, and was suitable for the determination of 4 furocoumarins in cosmetics.

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.

Occurrence and mass balance of isoflavones on an experimental grassland field.

Isoflavones and coumestrol (COU) are estrogenic compounds that are naturally produced by plants (e.g., red clover, soybeans). Although these compounds have been extensively studied in food and feed, only little is known about their environmental fate. Therefore, we investigated the occurrence of isoflavones (formononetin, daidzein, equol, biochanin A, and genistein) and COU over 3.5 years in red clover, manure, and soil of a grassland field with and without manure application, as well as their emission via drainage water. Isoflavones were regularly quantified in plant (≤ 15 × 10(6) ng/g(dry weight (dw))), manure (≤ 230 × 10(3) ng/g(dw)), soil (≤ 3.4 × 10(3) ng/g(dw)), and drainage water samples (≤ 3.6 × 10(3) ng/L). In contrast, COU was observed only in manure and soil. Cumulative isoflavone loads emitted via drainage water were around 0.2 × 10(-3) kg/ha/y, which is very little compared to the amounts present in red clover (105-220 kg/ha/y), manure (0.5-1.0 kg/ha/y), and soil (0.1-5.1 kg/ha/y). Under good agricultural practice, no additional emission of isoflavones into drainage water was observed after manure application. With calculated 17ß-estradiol equivalents up to 0.46 ng/L in drainage water, isoflavones can constitute a dominant and ecotoxicological relevant portion of the total estrogenicity in small rural river catchments.

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)).

LC analysis of coumestrol incorporated into topical lipid nanoemulsions.

A simple, rapid, and sensitive LC method to determine coumestrol incorporated in the lipid nanoemulsions was validated. The analyses were performed at room temperature on a reversed-phase C18 column using a mobile phase composed of methanol/water with 0.1% trifluoracetic acid (70:30, v/v) at 0.8 mL min(-1). The detection was carried out on a UV detector at 343 nm. The linearity, in the range of 0.1-6.0 microg/mL, presented a determination coefficient (r2) of 0.999, calculated by the least square method. No interferences of the oil core or the gelling excipients were detected. The R.S.D. values for intra- and inter-day precision experiments were lower than 2%. The recovery ranged from 99.42% to 100.72%. Finally, the proposed method was successfully applied to determine coumestrol incorporated in the proposed topical formulations.

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.

A Medicago truncatula mutant hyper-responsive to mycorrhiza and defective for nodulation.

One key strategy for the identification of plant genes required for mycorrhizal development is the use of plant mutants affected in mycorrhizal colonisation. In this paper, we report a new Medicago truncatula mutant defective for nodulation but hypermycorrhizal for symbiosis development and response. This mutant, called B9, presents a poor shoot and, especially, root development with short laterals. Inoculation with Glomus intraradices results in significantly higher root colonisation of the mutant than the wild-type genotype A17 (+20% for total root length, +16% for arbuscule frequency in the colonised part of the root, +39% for arbuscule frequency in the total root system). Mycorrhizal effects on shoot and root biomass of B9 plants are about twofold greater than in the wild-type genotype. The B9 mutant of M. truncatula is characterised by considerably higher root concentrations of the phytoestrogen coumestrol and by the novel synthesis of the coumestrol conjugate malonyl glycoside, absent from roots of wild-type plants. In conclusion, this is the first time that a hypermycorrhizal plant mutant affected negatively for nodulation (Myc(++), Nod (-/+) phenotype) is reported. This mutant represents a new tool for the study of plant genes differentially regulating mycorrhiza and nodulation symbioses, in particular, those related to autoregulation mechanisms.

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.

Glyphosate-tolerant alfalfa is compositionally equivalent to conventional alfalfa (Medicago sativa L.).

Glyphosate-tolerant alfalfa (GTA) was developed to withstand over-the-top applications of glyphosate, the active ingredient in Roundup agricultural herbicides. As a part of the safety assessment, GTA (designated J101 x J163) was grown under controlled field conditions at geographically diverse locations within the United States during the 2001 and 2003 field seasons along with control and other conventional alfalfa varieties for compositional assessment. Field trials were conducted using a randomized complete block design with four replication blocks at each site. Alfalfa forage was harvested at the late bud to early bloom stage from each plot at five field sites in 2001 (establishment year) and from four field sites in 2003 (third year of stand). The concentration of proximate constituents, fibers, amino acids, coumestrol, and minerals in the forage was measured. The results showed that the forage from GTA J101 x J163 is compositionally equivalent to forage from the control and conventional alfalfa varieties.

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.