Coumestrol Content Research Articles

Effect of gelling agents on properties of coumestrol-loaded nanoemulsions – Formulation, skin retention/permeation, antioxidant and skin photoprotective activities

Coumestrol is an isoflavonoid that exhibits antioxidant activity. In this study, hydrogels containing coumestrol-loaded nanoemulsions were designed to protect the skin against UVA/UVB light. The systems investigated were coumestrol-loaded nanoemulsions obtained by spontaneous emulsification procedure formulated in semisolid hydrogels composed of ionic (Carbopol 940®), cationic (chitosan) or non-ionic (hydroxyethylcellulose) gelling materials. Transmission electron microscopy images of formulations showed oil droplets exhibiting 200–300 nm. Hydrogels displayed non-Newtonian shear-thinning behavior. Coumestrol content in formulations remained close to 100 % (1 mg/mL) over time (up to 90 days). Regardless of the gelling material used, a lowering of coumestrol release rate was observed, in comparison with the release from nanoemulsion. The permeation and retention of coumestrol in porcine ear skin was evaluated using Franz-type diffusion cells. A higher retention of coumestrol in the epidermis was noticed (20 μg/g) from Carbopol 940-based formulation after 8 h of kinetics. No histological evidence of tissue damage was observed. The nanoemulsion containing coumestrol thickened with Carbopol 940® was selected for further studies. The formulation exhibited antioxidant potential as estimated by TRAP and TAR assays. The skin photoprotective effect was evidenced by means of TBARS, protein carbonylation, and protein thiol content experiments. Overall, our research suggests that the formulation is promising to protect skin against oxidative damage from UVA/UVB light.

Effects of infection with the viral complex alfalfa dwarf disease on productivity traits and coumestrol production in alfalfa plants

Context Viral infections generate yield losses in alfalfa due to the impact on growth, seed production, impaired nitrogen fixation and fungal disease susceptibility, but also stimulate production of coumestrol, which can exhibit oestrogenic activity in livestock and human. Aims We investigated the change in coumestrol content and its relationship with morphological and productivity traits in alfalfa plants infected with alfalfa viral complex. Methods Seven genotypes from cultivar Monarca SP-INTA and six of Traful PV-INTA were used. Plants were cloned and treated as non-infected and infected with alfalfa viral complex (alfalfa dwarf disease). Productivity and morphological traits were assessed. Disease symptoms were used to estimate the genotype severity index (GSI, %), coumestrol content was determined by high pressure liquid chromatography. Key results Viral infection reduced internode distance (iD) for the majority of Monarca genotypes, whereas in Traful one was affected, generating dwarfism. The iD reduction positively correlated with GSI only in Monarca genotypes. Coumestrol content varied between genotypes of each of the two cultivars. Monarca genotypes ranged between 350 and 1140 (infected and non-infected) ppm, and Traful genotypes ranged between 0 and 942 (infected and non-infected) ppm. However, viral infection promoted coumestrol increase in three genotypes only. Conclusions All Monarca genotypes studied seem to be more sensitive to viral infections than those of Traful, as GSI demonstrated. Independently of cultivar, coumestrol content increased for three genotypes. High coumestrol content was correlated with low GSI. Implications The knowledge of genotype response to viral infections would be useful for breeders to select superior genotypes as well as to originate new cultivars either as forage or as natural sources of phytoestrogens.

Sequential infections by 32 isolates of

Context Studies of Phoma black stem and leaf spot disease (caused by Phoma medicaginis) in annual medics (Medicago spp.) normally involve a ‘once-only’ inoculation not reflecting multiple pathogen infection and phytoestrogen production cycles in the field. Phytoestrogen production by plants can result in lower ovulation rates in grazing animals. Aims We aimed to determine whether sequential infections by P. medicaginis increase production of phytoestrogens in annual medics, and to measure the genetic diversity of isolates. Methods In a greenhouse experiment, pathogenicity and virulence were investigated across 32 isolates of P. medicaginis following one, two or three rounds of inoculation of M. polymorpha var. brevispina. Production of the phytoestrogens coumestrol and 4′-O-methyl coumestrol was measured, and correlation with disease parameters assessed. DNA sequencing using ITS, β-tubulin, calmodulin and P. medicaginis-specific EFNI-1α was applied for phylogenetic analysis of isolates from Western Australia and elsewhere. Key results Across isolates, highest leaf disease incidence was 76%, petiole disease incidence 61%, leaf disease severity 52% and petiole disease severity 53%. Stem coumestrol content range was 45–1247 mg kg−1, and 4′-O-methyl coumestrol 0–344 mg kg−1. All measures were highest after three rounds of inoculation. Overall, there was a positive correlation of leaf disease incidence with coumestrol content (P < 0.05) and of both leaf and petiole disease incidence with 4′-O-methylcoumestrol content (P < 0.01, P < 0.05, respectively). Phylogenetic analysis revealed a high degree of genetic similarity among Western Australian isolates, generally grouping into a single separate cluster across the four markers, and genetically distinct from isolates sourced outside Australia. Conclusions Leaf disease incidence was the best discriminating disease parameter for coumestrol and 4′-O-methylcoumestrol content. Western Australian isolates of P. medicaginis were genetically similar and unique, possibly due to geographic separation. Implications The study emphasised the importance of sequential inoculations when screening annual Medicago genotypes towards developing cultivars with superior disease resistance and enhanced animal reproductive outcomes.

Coumestrol content in alfalfabreeding populations

Aim. Alfalfa is a rich source of phytoestrogens, among them coumestrol which shows strong estrogenic activity that can adversely affect the health of domestic animals. The aim of the study was to determine the variation in coumestrol content in leaves of alfalfa breeding populations, present in the breeding program of Agricultural Institute Osijek in Croatia. Method. Twelve alfalfa populations were selected based on their high forage yield and good persistence. Coumestrol was extracted using acidifi ed methanol as an organic solvent from lyophilized and ground alfalfa leaves, while for detection and quantifi cation was used. Results. Signifi cant differences were observed between the studied populations with average coumestrol content of 435.67 mg/kg of dry matter (DM). The highest content of coumestrol was determined in breeding population Rs-21 (619.53 mg/kg of DM). Conclusions. Populations Rs-33 and Rs-20 had the lowest coumestrol content (82.18 and 86.58 mg/kg, respectively) and present a potential breeding source for creat- ing new contemporary cultivars with decreased coumestrol content

Study of hypocholesterolemic and antiatherosclerotic properties of Medicago sativa L. cultivated in Egypt

Alfalfa was reported to be hypocholesterolemic and antiatherosclerotic. Saponin glycosides were suggested to be responsible for this activity by neutralizing cholesterol in the stomach, enabling it to be excreted from the body. This makes alfalfa superior to the current anti-cholesterol medications which act by blocking cholesterol synthesizing apparatus. This study was undertaken in an attempt to prepare an alfalfa extract with high saponin content and free or low content of other toxic constituents found in alfalfa (canavanine and coumestrol), which causes serious side effects. A patent process was used to render alfalfa toxin free and to produce a drug present in the international market. Our study is based on monitoring the Egyptian crop at two different localities for its saponin, canavanine and coumestrol contents at different growth stages. The study showed that the tested extract of the chosen stage to be biologically studied (hypocholesterolemic and antiatherosclerotic effects) on the basis of the highest saponin content was just before fruiting stage of the locality A. This stage was free from both coumestrol and canavanine. The study proved that the Egyptian crop of alfalfa was found to safely reduce natural cholesterol and to possess a strong antiatherosclerotic activity. This extract (1) produced the most significant decrease in total cholesterol and LDL-cholesterol by 85.1 and 88%, respectively, of the corresponding levels in hypercholesterolemic rabbits. This decrease is more significant than that produced by gemfibrozil (73 and 74%) upon concomitant administration with a cholesterol enriched diet using the same animal model at the tested dose level. Also, it was obvious that all alfalfa preparations produced significant antioxidant properties. All alfalfa extracts possessed antiatherosclerotic activity as observed by the almost normalization of the aortic sections upon concomitant use of alfalfa extracts with cholesterol-enriched diet.

Comprehensive RNA sequencing and co-expression network analysis to complete the biosynthetic pathway of coumestrol, a phytoestrogen

Coumestrol (CMS), a coumestan isoflavone, plays key roles in nodulation through communication with rhizobia, and has been used as phytoestrogens for hormone replacement therapy in humans. Because CMS content is controlled by multiple genetic factors, the genetic basis of CMS biosynthesis has remained unclear. We identified soybean genotypes with consistently high (Daewonkong) or low (SS0903-2B-21-1-2) CMS content over 2 years. We performed RNA sequencing of leaf samples from both genotypes at developmental stage R7, when CMS levels are highest. Within the phenylpropanoid biosynthetic pathway, 41 genes were tightly connected in a functional co-expression gene network; seven of these genes were differentially expressed between two genotypes. We identified 14 candidate genes involved in CMS biosynthesis. Among them, seven were annotated as encoding oxidoreductases that may catalyze the transfer of electrons from daidzein, a precursor of CMS. Two of the other genes, annotated as encoding a MYB domain protein and a MLP–like protein, may increase CMS accumulation in response to stress conditions. Our results will help to complete our understanding of the CMS biosynthetic pathway, and should facilitate development of soybean cultivars with high CMS content that could be used to promote the fitness of plants and human beings.

Correction: The number of measurements needed to obtain high reliability for traits related to enzymatic activities and photosynthetic compounds in soybean plants infected with Phakopsora pachyrhizi.

[This corrects the article DOI: 10.1371/journal.pone.0192189.].

Prediction of coumestrol content in unirrigated lucerne crops using weather variables

ABSTRACTElevated coumestrol levels in lucerne (Medicago sativa L.) can reduce the reproductive performance of ewes. A model to predict coumestrol content of lucerne between summer and late autumn was developed to assess the risk of a crop having elevated levels. Data from previous studies have shown fungal damage to be the major contributor to coumestrol levels, but the fungal infestation levels that can cause a risk to reproduction in sheep are difficult to quantify. A number of variables were tested and the sum of rainfall and days where relative humidity was over 95% at 0900 h was found to have the best fit with coumestrol content of lucerne crops. The model was then tested with independent data from the literature and field. A decision tree is proposed as a practical user-friendly guide to predict whether coumestrol content is above or below a risk threshold of 25 mg/kg DM.

Alfalfa Coumestrol Content in Response to Development Stage, Fungi, Aphids, and Cultivar

Core Ideas Alfalfa cultivars still produce coumestrol contents detrimental to ewe reproduction.Coumestrol increased simultaneously across alfalfa of different development stages.Coumestrol related to fungal symptoms but prediction from symptoms was inaccurate.Coumestrol did not respond to pea aphids as severely as previously reported. Coumestrol produced by alfalfa (Medicago sativa L.) can reduce the ovulation rate of ewes. This study isolated agronomic factors that affect coumestrol levels in alfalfa. Fungal diseases explained most differences in coumestrol. Alfalfa inoculated with Stemphylium vesicarium contained 169 ± 25.1 mg kg−1 dry matter (DM) compared with 3.4 ± 0.84 mg kg−1 DM in controls. However, in field‐grown alfalfa there was a low relationship (R2 = 0.388) between coumestrol content and visual fungal damage score which indicates a need for alternative methods of prediction. In the field, a range of cultivars all reached coumestrol levels reported to pose a risk to ewe reproductive performance (>25 mg kg−1 DM). In S. vesicarium‐inoculated leaves, the modern cultivar ‘Stamina 5’ had 396 ± 82.4 mg kg−1 DM compared with the 40‐yr‐old industry standard ‘Wairau’ at 143 ± 35.6 mg kg−1 DM. Flowering in isolation did not produce a coumestrol response, with increases detected or not detected simultaneously in vegetative and flowering plants. Pea aphids had a minor effect on coumestrol content. When alfalfa was subjected to ∼5 aphids per stem for 4 wk coumestrol increased from 2.4 ± 0.39 to 5.3 ± 0.65 mg kg−1 DM. This study highlights fungal pathogens traditionally not considered problematic in alfalfa stands due to limited effects on stand persistence and yield, as important causal agents of elevated coumestrol levels. Plant breeding to reduce their impact is recommended.

The number of measurements needed to obtain high reliability for traits related to enzymatic activities and photosynthetic compounds in soybean plants infected with Phakopsora pachyrhizi.

Asian rust affects the physiology of soybean plants and causes losses in yield. Repeatability coefficients may help breeders to know how many measurements are needed to obtain a suitable reliability for a target trait. Therefore, the objectives of this study were to determine the repeatability coefficients of 14 traits in soybean plants inoculated with Phakopsora pachyrhizi and to establish the minimum number of measurements needed to predict the breeding value with high accuracy. Experiments were performed in a 3x2 factorial arrangement with three treatments and two inoculations in a random block design. Repeatability coefficients, coefficients of determination and number of measurements needed to obtain a certain reliability were estimated using ANOVA, principal component analysis based on the covariance matrix and the correlation matrix, structural analysis and mixed model. It was observed that the principal component analysis based on the covariance matrix out-performed other methods for almost all traits. Significant differences were observed for all traits except internal CO2 concentration for the treatment effects. For the measurement effects, all traits were significantly different. In addition, significant differences were found for all Treatment x Measurement interaction traits except coumestrol, chitinase and chlorophyll content. Six measurements were suitable to obtain a coefficient of determination higher than 0.7 for all traits based on principal component analysis. The information obtained from this research will help breeders and physiologists determine exactly how many measurements are needed to evaluate each trait in soybean plants infected by P. pachyrhizi with a desirable reliability.

Perennial legumes as a source of ingredients for healthy food: proximate, mineral and phytoestrogen composition and antibacterial activity.

Perennial legumes have been used as edible or medicinal plants since ancient times. The focus of the current study are perennial legumes-Trifolium pratense L., T. medium L., Medicago sativa L., M. lupulina L., Onobrychis viciifolia Scop., Astragalus glycyphyllos L. and A. cicer L.-of branching stage as a potential source of value-added ingredients for healthy food. Freeze-dried samples were analysed for proximal composition, mineral, isoflavone and coumestrol contents as well as for antimicrobial activity. Legumes were protein-rich (23.0/100g on average). Mineral contents in 100g of plant dry matter averaged: K 2.64g, Ca 1.81g, Mg 0.475g, P 0.324g, Zn 2.76mg and Fe 37.8mg. According to the total amount of phytoestrogens, the species ranked as follows: T. medium (34.4mg/g)≫T. pratense≫O. viciifolia≥M. sativa=A. cicer=M. lupulina≥A. glycyphyllos (0.207mg/g). Extracts of legumes, especially that of O. viciifolia, exhibited noticeable potency to inhibit the growth of Gram-positive and Gram-negative bacteria. Perennial legumes of branching stage can be used as protein, mineral and phytoestrogen rich source for food ingredients and supplements.

Lucerne with a high coumestrol content is not a suitable feed for ewes just before and during the early breeding period

Abstract The objective was to determine whether grazing lucerne for 7 days before breeding and for the first 7 days of breeding affected the reproductive performance of ewes with different body condition scores (average of 3.2 versus 3.7). This was compared with grazing a grass-based sward with a high proportion of dead herbage that was available at the time of breeding in a summer dry region (Wairarapa) in autumn 2016. The lucerne contained large amounts of fungal infected stem and dead herbage, which induced high levels of coumestrol (107 ± 15 mg/kg DM). By the end of the feeding period the grass and lucerne on offer were both of low quality (7.8 MJ ME/kg DM). Ewes that grazed lucerne for 7 days before breeding and for the first 7 days of the breeding period produced 18 fewer (P

Non-isoflavone phytoestrogenic compound contents of various legumes

Widely consumed legumes including chickpeas, red kidney beans, haricot beans, yellow lentils, red lentils and green lentils were analysed to determine the content of non-isoflavone phytoestrogenic compounds such as quercetin, rutin, apigenin, coumestrol and lignan (matairesinol and secoisolariciresinol). Methanolic extracts obtained by ultrasound-assisted extraction were analysed by the triple quadrupole LC–MS/MS. Red kidney beans were the best source of quercetin (603.2 ± 307.2 μg/kg) and rutin (73.4 ± 14.0 μg/kg). Apigenin and secoisolariciresinol contents were the highest in yellow lentils (18.5 ± 0.84 μg/kg) and haricot beans (451.9 ± 192.2 μg/kg), respectively. Coumestrol contents of haricot beans (18.5 ± 1.45 μg/kg) and red kidney beans (18.5 ± 1.26 μg/kg) were equal to each other, and these were determined as the highest coumestrol content values. The best sources of matairesinol occurred in green lentils (28.2 ± 0.18 μg/kg) and chickpeas (27.7 ± 1.83 μg/kg). Differences between contents of each sample of the same legume were significant and remarkable, especially for quercetin and secoisolariciresinol.

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.

Phytoestrogen Content of Cereals and Cereal-Based Foods Consumed in the UK

Dietary phytoestrogens may be involved in the occurrence of chronic diseases. Reliable information on the phytoestrogen content in foods is required to assess dietary exposure and disease risk in epidemiological studies. However, there is little information on isoflavone, lignan, and coumestrol content of cereals and cereal-based foods, leading to an underestimation of intake. This is the first study of phytoestrogens (isoflavones: biochanin A, daidzein, formononetin, genistein, glycitein; lignans: matairesinol, secoisplariciresinol; coumestrol) in a comprehensive selection of 101 cereals and cereal-based foods—including breads, breakfast cereals, biscuits, pasta and rice—consumed in the UK using a sensitive LCMS technique with 13 C-labelled internal standards. Phytoestrogens were detected in all foods analyzed; bread contained the highest amount of phytoestrogens—many as isoflavones—with an average content of 375 ± 67 μ g/100 g wet weight (excluding soya-linseed bread with 12,000 μ g/100 g). Most other foods contained less than 100 μ g/100 g, many as lignans. Our study shows that all foods analyzed contained phytoestrogens, with the highest amount found in breads, making them one of the main sources of dietary phytoestrogens in the UK. These results will allow a more accurate estimation of exposure to dietary phytoestrogens.

Coumestrol content of alfalfa following ensilage

An experiment evaluated the influence of several variables on the coumestrol content of alfalfa ( Medicago sativa) silages. Two cultivars of alfalfa (Morava and Palava) were each harvested on May, June, and October 2000. The forage was allowed to wilt in the swath to approximately 300 or 500 g/kg dry matter (DM) content and then it was ensiled without further treatment. The number of lactic bacteria, mold, and yeast as well as pH and concentration of lactic acid were indicative of adequate preservation. The majority of the initial content of coumestrol disappeared during 50 days of ensiling, but some was still detected after 150 days. The coumestrol concentration in silages varied with both cultivar and maturity of plant while there was no significant effect of the dry matter content of feed forage. This report shows that wilting in the swath caused remarkable increase in coumestrol content of alfalfa.

Coumestrol content of alfalfa following ensilage

An experiment evaluated the influence of several variables on the coumestrol content of alfalfa ( Medicago sativa ) silages. Two cultivars of alfalfa (Morava and Palava) were each harvested on May, June, and October 2000. The forage was allowed to wilt in the swath to approximately 300 or 500 g/kg dry matter (DM) content and then it was ensiled without further treatment. The number of lactic bacteria, mold, and yeast as well as pH and concentration of lactic acid were indicative of adequate preservation. The majority of the initial content of coumestrol disappeared during 50 days of ensiling, but some was still detected after 150 days. The coumestrol concentration in silages varied with both cultivar and maturity of plant while there was no significant effect of the dry matter content of feed forage. This report shows that wilting in the swath caused remarkable increase in coumestrol content of alfalfa.

Virus infection stimulates phyto‐oestrogen production in pasture legume plants growing in grazed swards

SummaryFour field experiments were sown with AMV‐infected or healthy seed of burr medic (Medicago polymorpha) and grazed by sheep; two were sown with cv. Circle Valley and two with cv. Santiago. Seed‐infected plants acted as primary sources for virus spread by naturally occurring aphids. Insecticides and admixture with annual ryegrass (Lolium rigidum), a non‐host of AMV, suppressed virus spread to different extents in the plots sown with infected seed. Effects of the different amounts of virus spread obtained on overall concentration of the oestrogenic compound coumestrol (dihydroxycoumestan) in dry stems and pods, and on seed production were measured in the medic. With cv. Santiago, stem and pod coumestrol concentration values for plots sown with healthy seed were significantly smaller than those for all plots sown with infected seed regardless of whether they were sprayed. With cv. Circle Valley, the coumestrol values for stems from plots sown with healthy seed were significantly smaller than those for unsprayed plots sown with infected seed but not than those for sprayed plots or ones with grass admixtures, and there were no significant differences with pods. There was always a significant positive relationship between concentration of coumestrol in medic stems and percentage AMV infection of swards; this was also so with pods in two experiments. A linear model best fitted this relationship with cv. Circle Valley but a logarithmic model did so with cv Santiago. In glasshouse grown plants, the coumestrol content of dried medic shoots was increased 11 (cv. Circle Valley) and five (cv. Santiago) times by AMV infection. AMV increased mean coumestrol concentrations up to 256 ppm (field) and 237 ppm (glasshouse) in stems and 223 ppm in pods (field). Sowing healthy seed in new pasture swards was an effective strategy for minimising coumestrol accumulation in burr medic swards. Two, but not single, applications of a newer generation pyrethroid insecticide to swards in which AMV was spreading significantly diminished coumestrol accumulation but applying organophosphorus insecticide twice and carbamate insecticide repeatedly did not. Medic seed yields and individual seed weights were sometimes significantly increased by the treatments, suppression of AMV spread by regular carbamate sprays being sufficient to increase seed yield by 55%.

Production of flavonoids by Psoralea hairy root cultures

Eighteen transformed root cultures from 7 Psoralea plant species (Leguminosae) were established with the objective of producing daidzein and related flavonoids. All the 18 hairy root lines grew fast and had the same capacities for biomass production. Each of them produced daidzein as an intracellular secondary metabolite. The Lach5 hairy root line, obtained from P. lachnostachys, was a high producing line for daidzein and was further studied for biomass and flavonoid production. This root line showed exponential growth. Chitosan was used for elicitation purposes as well as for its permeabilizing effect. Little elicitation effect could be demonstrated and the metabolite release in the medium was weak (about 1%) and limited to the first 29 h after chitosan addition. Daidzein was demonstrated to be more concentrated in young parts (apexes) whereas coumestrol content was higher in older parts (brown tissues). Compared to callus cultures from the same plant species, hairy roots displayed comparable concentrations. However, high-producing lines were more frequently found with hairy roots (4 out of 18) than with callus cultures (4 out of 217)

Morphologische und agronomische Merkmale autochthoner annueller Medicago‐Arten in Marokko

AbstractMorphological and agronomic characteristics of autochthonous annual Medkago species in MoroccoAutochthonous annual Medicago species were collected on non‐alkaline soils and evaluated as to their potential agricultural use. 411 Medicago ecotypes were found at 108 sites. With increasing rainfall and altitude of collecting sites, a decrease was recorded in the length of sprouts, in sward height, in leaf size, and in dry matter yield. High rainfall regions were found to select late flowering ecotypes. All Medicago ecotypes tested showed broad intraspecific variation in seed yield, leaf size and seed weight. Coumestrol content of leaves at flowering was generally low; on average 115 ppm of dry matter. No relationship was found between collection sites and coumestrol content. In a field trial comparing 112 ecotypes with nine introduced cultivars at two contrasting sites, thirty ecotypes proved to be better adapted to local conditions than the cultivars.