DESIGNER fraction concept unmasks minor bioactive constituents in red clover (Trifolium pratense L.).

In botanical extracts, highly abundant constituents can mask or dilute the effects of other, and often, more relevant biologically active compounds. To facilitate the rational chemical and biological assessment of these natural products with wide usage in human health, we introduced the DESIGNER approach of Depleting and Enriching Selective Ingredients to Generate Normalized Extract Resources. The present study applied this concept to clinical Red Clover Extract (RCE) and combined phytochemical and biological methodology to help rationalize the utility of RCE supplements for symptom management in postmenopausal women. Previous work has demonstrated that RCE reduces estrogen detoxification pathways in breast cancer cells (MCF-7) and, thus, may serve to negatively affect estrogen metabolism-induced chemical carcinogenesis. Clinical RCE contains ca. 30% of biochanin A and formononetin, which potentially mask activities of less abundant compounds. These two isoflavonoids are aryl hydrocarbon receptor (AhR) agonists that activate P450 1A1, responsible for estrogen detoxification, and P450 1B1, producing genotoxic estrogen metabolites in female breast cells. Clinical RCE also contains the potent phytoestrogen, genistein, that downregulates P450 1A1, thereby reducing estrogen detoxification. To identify less abundant bioactive constituents, countercurrent separation (CCS) of a clinical RCE yielded selective lipophilic to hydrophilic metabolites in six enriched DESIGNER fractions (DFs 01-06). Unlike solid-phase chromatography, CCS prevented any potential loss of minor constituents or residual complexity (RC) and enabled the polarity-based enrichment of certain constituents. Systematic analysis of estrogen detoxification pathways (ERα-degradation, AhR activation, CYP1A1/CYP1B1 induction and activity) of the DFs uncovered masked bioactivity of minor/less abundant constituents including irilone. These data will allow the optimization of RCE with respect to estrogen detoxification properties. The DFs revealed distinct biological activities between less abundant bioactives. The present results can inspire future carefully designed extracts with phytochemical profiles that are optimized to increase in estrogen detoxification pathways and, thereby, promote resilience in women with high-risk for breast cancer. The DESIGNER approach helps to establish links between complex chemical makeup, botanical safety and possible efficacy parameters, yields candidate DFs for (pre)clinical studies, and reveals the contribution of minor phytoconstituents to the overall safety and bioactivity of botanicals, such as resilience promoting activities relevant to women's health.

Effects of forage and grain legume-based silages supplemented with faba bean meal or rapeseed expeller on lactational performance, nitrogen utilization, and plasma amino acids in dairy cows.

The objective of this experiment was to investigate the effect of forage type [red clover (51%)-grass silage, i.e., RCG; vs. faba bean (66%)-grass silage, i.e., FBG] and concentrate type (faba bean, FB; vs. rapeseed expeller, RE) on lactational performance, milk composition and nitrogen (N) utilization in lactating dairy cows. Eight lactating multiparous Nordic Red cows were used in a replicated 4 × 4 Latin Square experiment, with 21-d periods, in a 2 × 2 factorial arrangement of treatments. The experimental treatments were as follows: (1) RCG with RE, (2) RCG with FB, (3) FBG with RE, and (4) FBG with FB. Inclusion rates of RE and FB were isonitrogenous. Crude protein contents of the experimental diets were 16.3, 15.9, 18.1, and 17.9% of dry matter, respectively. All diets included oats and barley and were fed ad libitum as total mixed rations with forage-to-concentrate ratio of 55:45. Dry matter intake and milk yield were recorded daily, and spot samples of urine, feces, and blood were collected at the end of each experimental period. Dry matter intake did not differ across diets, averaging 26.7 kg/d. Milk yield averaged 35.6 kg/d and was 1.1 kg/d greater for RCG versus FBG, and milk urea N concentration was lower for RCG compared with FBG. Milk yield was 2.2 kg/d and milk protein yield 66 g/d lower for FB versus RE. Nitrogen intake, urinary N, and urinary urea N excretions were lower, and milk N excretion tended to be lower for RCG compared with FBG. The proportion of the dietary N excreted as fecal N was larger in cows fed RCG than for those fed FBG, and the opposite was true for urinary N. We detected an interaction for milk N as percentage of N intake: it increased with RE compared with FB for RCG-based diet, but only a marginal increase was observed for FBG-based diet. Plasma concentration of His and Lys were lower for RCG than for FBG, whereas His tended to be greater and Lys lower for FB compared with RE. Further, plasma Met concentration was around 26% lower for FB than for RE. Of milk fatty acids, saturated fatty acids were decreased by RCG and increased by FB compared with FBG and RE, respectively, whereas monounsaturated fatty acids were increased by RCG versus FBG, and were lower for FB than for RE. In particular, 18:1n-9 concentration was lower for FB compared with RE. Polyunsaturated fatty acids, such as 18:2n-6 and 18:3n-3, were greater for RCG than for FBG, and 18:2n-6 was greater and 18:3n-3 was lower for FB versus RE. In addition, cis-9,trans-11 conjugated linoleic acid was lower for FB compared with RE. Faba bean whole-crop silage and faba bean meal have potential to be used as a part of dairy cow rations, but further research is needed to improve their N efficiency. Red clover-grass silage from a mixed sward, without inorganic N fertilizer input, combined with RE, resulted in the greatest N efficiency in the conditions of this experiment.

Antioxidant properties of Trifolium resupinatum and its therapeutic potential for Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) is the most common cause of dementia and is characterized by a progressive deterioration in cognitive function, which typically begins with impairment in memory. Persian clover (Trifolium resupinatum) is an annual plant found in central Asia. Due to its contents (high flavonoid and isoflavones), extensive researches have been done on its therapeutic properties, such as multiple sclerosis (MS) treatment. In this study, we investigate the neuroprotective effects of this plant on Streptozotocin (STZ)-induced AD in rats. MATERIAL AND METHODS: This research aimed to evaluate the neuroprotective effect of Trifolium resupinatum on the spatial learning and memory, superoxide dismutase (SOD), expressions of ß amyloid 1-42 (Ab 1-42 ), and b amyloid 1-40 (Ab 1-40 ) in the hippocampus of STZ-induced Alzheimer rats. RESULTS: Our data showed that Trifolium resupinatum extract administration for two weeks before and one week after AD induction significantly improves maze escape latency ( p = 0.027, 0.001 and 0.02 in 100, 200, and 300 mg of the extract, respectively) and maze retention time ( p = 0.003, 0.04 and 0.001 in 100, 200, and 300 mg of the extract, respectively). Also, the administration of this extract significantly increases the SOD levels from 1.72+0.20 to 2.31+0.45 ( p = 0.009), 2.48+0.32 ( p = 0.001) and 2.33+0.32 ( p = 0.007) and decreases the expressions of Ab 1-42 ) ( p = 0.001 in all concentrations of the extract) and Ab 1-40 ) ( p = 0.001 in all concentrations of the extract) in the rat's hippocampus. CONCLUSIONS: This study suggests that the alcoholic extract of Trifolium resupinatum has anti-Alzheimer and neuroprotective effects on rats.

Red clover (Trifolium pratense L.) extract inhibits ferroptotic cell death by modulating cellular iron homeostasis.

ETHNOPHARMACOLOGICAL RELEVANCE: Red clover (Trifolium pratense L.) is a traditional Chinese medicine and use as herbal medicine which has the effects of regulating menopausal symptoms, heart problem, inflammatory disease, psoriasis and cognitive deficits. In previous reported, the studies of red clover were mainly focused on clinical practice. the pharmacological functions of red clover not fully elucidated. AIM OF THE STUDY: To identify the molecules that regulate ferroptosis, we examined whether red clover (Trifolium pratense L.) extracts (RCE) affected ferroptosis induced by chemical treatment or cystine/glutamate antiporter (xCT) deficiency. MATERIALS AND METHODS: Cellular models for ferroptosis were induced by erastin/Ras-selectiv lethal 3 (RSL3) treatment or xCT deficiency in mouse embryonic fibroblasts (MEFs). Intracellular iron and peroxidized lipid levels were determined using Calcein-AM and BODIPY-C11 fluorescence dyes, respectively. Protein and mRNA were quantified by Western blot and real-time polymerase chain reaction, respectively. RNA sequencing analysis was performed on xCT-/- MEFs. RESULTS: RCE significantly suppressed ferroptosis induced by both erastin/RSL3 treatment and xCT deficiency. The anti-ferroptotic effects of RCE correlated to ferroptotic phenotypic changes such as cellular iron accumulation and lipid peroxidation in cellular ferroptosis models. Importantly, RCE affected levels of iron metabolism-related proteins including iron regulatory protein 1, ferroportin 1 (FPN1), divalent metal transporter 1, and transferrin receptor. RNA sequencing analysis of xCT-/- MEFs identified that expression of cellular defense genes was upregulated, while expression of cell death-related genes was downregulated, by RCE. CONCLUSION: RCE potently suppressed ferroptosis triggered both by erastin/RSL3 treatment and xCT deficiency by modulating cellular iron homeostasis. This is the first report that RCE has therapeutic potential in diseases associated with ferroptotic cell death, particularly ferroptosis induced by dysregulation of cellular iron metabolism.

Effects of exogenous taurine on growth, photosynthesis, oxidative stress, antioxidant enzymes and nutrient accumulation by Trifolium alexandrinum plants under manganese stress.

Plants essentially require manganese (Mn) for their normal metabolic functioning. However, excess Mn in the cellular environment is detrimental to plant growth, development, and physio-biochemical functions. Taurine (TAU) is an amino acid with potent antioxidant and anti-inflammatory properties in animals and humans. However, no previous study has investigated the potential of TAU in plant metal stress tolerance. The current study provides some novel insights into the effect of TAU in modulating the defense system of Trifolium alexandrinum plants under Mn toxicity. Manganese toxicity resulted in higher oxidative stress and membrane damage through increased superoxide radical, hydrogen peroxide, malondialdehyde, and methylglyoxal generation alongside enhanced lipoxygenase (LOX) activity. Mn toxicity also resulted in limited uptake of potassium (K+), phosphorus (P), calcium (Ca2+), and increased the accumulation of Mn in both leaf and roots. However, TAU circumvented the Mn-induced oxidative stress by upregulating the activities of antioxidant enzymes (ascorbate peroxidase, peroxidase, catalase, glutathione reductase, glutathione-S-transferase, and superoxide dismutase) and levels of ascorbic acid, proline, anthocyanins, phenolics, flavonoids and glutathione (GSH). Taurine conspicuously improved the growth, photosynthetic pigments, hydrogen sulphide (H2S), and nitric oxide (NO) levels of Mn stressed plants. Taurine also improved the uptake of K+, Ca2+, P and reduced the Mn content in stressed plants. Overall, exogenous taurine might be a suitable strategy to combat Mn stress in T. alexandrinum plants but applications at field levels for various crops and metal toxicities and economic suitability need to be addressed before final recommendations.

Antidiabetic efficacy of Trifolium alexandrinum extracts hesperetin and quercetin in ameliorating carbohydrate metabolism and activating IR and AMPK signaling in the pancreatic tissues of diabetic rats.

Diabetes is a metabolic disease that is mainly characterized by hyperglycemia. The present work investigated the efficacy of the flavanones hesperetin (HES) and quercetin (Q) extracted from Trifolium alexandrinum (TA) to treat type 2 diabetic rats. Wistar albino rats were supplemented with a high fat diet (HFD) for 2 weeks and then administered streptozotocin to induce diabetes. Diabetic rats were orally treated with Q, HES, and TA extract at concentrations of 40, 50, and 200 mg/kg BW, respectively, for 4 weeks. Various biochemical, molecular, and histological analysis were performed to evaluate the antidiabetic effects of these treatments. Q, HES, and TA extract treatments all significantly improved diabetic rats' levels of serum glucose, insulin, glucagon, liver function enzymes, hepatic glycogen, α-amylase, lipase enzymes, lipid profiles, oxidative stress indicators, and antioxidant enzymes as compared with control diabetic untreated rats. In addition, supplementation with Q, HES, and TA extract attenuated the activities of glucose-6-phosphate; fructose-1,6-bisphospahate; 6-phosphogluconate dehydrogenase; glucose-6-phosphate dehydrogenase; glucokinase; and hexokinase in pancreatic tissue, and they improved the levels of glucose transporter 2 and glucose transporter 4. Furthermore, these treatments modulated the expressions levels of insulin receptor (IR), phosphoinositide 3-kinase (PI3K), AMP-activated protein kinase (AMPK), caspase-3, and interleukin-1ß (IL-1ß). Enhancement of the histological alterations in pancreatic tissues provided further evidence of the ability of Q, HES, and TA extract to exert antidiabetic effects. Q, HES, and TA extract remedied insulin resistance by altering the IR/PI3K and AMPK signaling pathways, and they attenuated type 2 diabetes by improving the antioxidant defense system.

Trifolium pratense extract increases testosterone and improves sperm characteristics and antioxidant status in diabetic rats.

Male reproductive dysfunction is a common complication of diabetes mellitus. Trifolium pratense exhibits antioxidant and antidiabetic effects. We investigated the effects of an extract of T. pratense on serum antioxidant status, sperm characteristics, testicular tissue changes and testosterone level in diabetic rats. Male Wistar rats were divided into six groups: 1, untreated control; 2, diabetic; 3 and 4, 100 or 200 mg/kg T. pratense extract treated, respectively; 5 and 6, diabetic 100 or 200 mg/kg T. pratense extract treated, respectively. Diabetes was induced by intraperitoneal injection of streptozotocin. After 3 weeks, serum glucose, testosterone and nitric oxide (NO); sperm parameters; testicular histology and total antioxidant capacity (TAC) were evaluated. In diabetic rats treated with T. pratense extract, sperm motility, count and viability, as well as TAC and testosterone were increased significantly compared to untreated diabetic rats, while serum NO and bcl-2 and p53 expression was decreased significantly compared to untreated diabetic rats. T. pratense extract reduced testicular tissue destruction caused by diabetes.

Trifolium pratense ethanolic extract alters the gut microbiota composition and regulates serum lipid profile in the ovariectomized rats.

BACKGROUND: Trifolium pratense (red clover) ethanolic extract (TPEE) has been used as a popular over-the-counter remedy for the management of menopausal symptoms. Prolonged consumption of herbal extract has been shown to regulate the composition of gut microbiota. This study was designed to elucidate the influence of TPEE on the gut microbiota composition in the ovariectomized (OVX) rats. METHODS: OVX rats were treated with TPEE at 125, 250, 500 mg/kg/day, or controls (pomegranate extract, 500 mg/kg/day; estradiol, 25 µg/kg/day) for 12 weeks. Gut microbiota analysis was conducted by extracting the microbial DNA from fecal samples and microbiome taxonomic profiling was carried out by using next-generation sequencing. The levels of serum biomarkers were analyzed using enzyme-linked immunosorbent assay (ELISA) kit. The prediction of functional biomarker of microbiota was performed using PICRUSt to investigate the potential pathways associated with gut health and serum lipid profile regulation. To study the correlation between gut microbiota composition and serum lipid levels, Spearman's correlation coefficients were defined and analyzed. Additionally, gas chromatography-mass spectrometry analysis was conducted to uncover additional physiologically active ingredients. RESULTS: TPEE-treated OVX rats showed significant reduction in serum triglycerides (TG), total cholesterols (TCHOL), and LDL/VLDL levels but increase in HDL level. The alteration in the pathways involve in metabolism was the most common among the other KEGG categories. Particularly, TPEE also significantly reduced the relative abundance of sequences read associated with inflammatory bowel disease (IBD) and the peroxisome proliferator-activated receptor (PPAR) signalling pathway. TPEE intervention was seen to reduce the Firmicutes to Bacteroidetes (F/B) ratio in the OVX rats, denoting a reduction in microbial dysbiosis in the OVX rats. Correlation analysis at the phylum level revealed that Bacteriodetes and Proteobacteria were strongly correlated with serum TG, TCHOL and HDL levels. At the species level, Bifidobacterium pseudolongum group was seen to positively correlate with serum HDL level and negatively correlated with serum AST, ALT, LDL/VLDL, TCHOL, and TG levels. CONCLUSIONS: TPEE treatment showed therapeutic benefits by improving the intestinal microbiota composition which strongly correlated with the serum lipid and cholesterol levels in the OVX rats.

Antimicrobial and Antioxidant Secondary Metabolites from Trifolium baccarinii Chiov. (Fabaceae) and Their Mechanisms of Antibacterial Action.

The treatment of infectious diseases with antimicrobial agents continues to present problems in modern-day medicine with many studies showing significant increase in the incidence of bacterial resistance to several antibiotics. The screening of antimicrobial activity of plant extracts and natural products has shown that medicinal plants are made up of a potential source of new anti-infective agents. The aim of this study was to evaluate the antimicrobial and antioxidant activities of extracts and compounds from the whole plant Trifolium baccarinii Chiov. and to determine their modes of antibacterial action. The plant extracts were prepared by maceration in organic solvents. The antimicrobial activities were evaluated using the broth microdilution method. The antioxidant activity was evaluated using the 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) assays. The mechanisms of antibacterial action were determined by lysis, salt tolerance assays, and antioxidant enzyme activities. The cytotoxic effect on the erythrocytes was determined by a spectrophotometric method. Biochanin A, formononetin, luteolin, luteolin-4'-O-ß-D-glucopyranoside, 4,7,2'-trihydroxy-4'-methoxyisoflavanol, sissotrin, 1-methyl-ß-D-glucopyranoside, ononin, D-mannitol, and 3-O-ß-D-glucuronopyranosylsoyasapogenol B were isolated from Trifolium baccarinii. The MeOH, EtOAc, and n-BuOH extracts as well as biochanin A, formononetin, luteolin, luteolin-4'-O-ß-D-glucopyranoside, 4,7,2'-trihydroxy-4'-methoxyisoflavanol, and sissotrin from Trifolium baccarinii displayed the highest antimicrobial and antioxidant activities. The MeOH extract and 4,7,2'-trihydroxy-4'-methoxyisoflavanol exhibited antibacterial activity through the bacteriolytic effect and reduction of the antioxidant defenses in the bacterial cells. The present study portrays Trifolium baccarinii as a potential natural source of antibacterial, antifungal, and antioxidant agents.

Comparison of the Polyphenolic Profile of Medicago sativa L. and Trifolium pratense L. Sprouts in Different Germination Stages Using the UHPLC-Q Exactive Hybrid Quadrupole Orbitrap High-Resolution Mass Spectrometry.

Identification and quantification of polyphenols in plant material are of great interest since they make a significant contribution to its total bioactivity. In the present study, an UPLC-Orbitrap-MS/MS approach using the variable data acquisition mode (vDIA) was developed and applied for rapid separation, identification, and quantification of the main polyphenolic compounds in Medicago sativa L. and Trifolium pratense L. sprouts in different germination stages. Based on accurate MS data and fragment ions identification strategy, a total of 29 compounds were identified by comparing their accurate masses, fragment ions, retention times, and literatures. Additionally, a number of 30 compounds were quantified by comparing to the reference standards. Data were statistically analysed. For both plant species, the sprouts of the third germination day are valuable sources of bioactive compounds and could be used in phytotherapy and nutrition. Although Trifolium pratense L. (Red Clover) is considered to be a reference for natural remedies in relieving menopause disorders, alfalfa also showed a high level of biological active compounds with estrogenic activity.

Trifolium pratense (Red Clover) Improve SIRT1 Expression and Glycogen Content in High Fat Diet-Streptozotocin Induced Type 2 Diabetes in Rats.

Flowering tops of Trifolium pratense L. (Fabaceae) are known for its traditional medicinal values. In present study, our aim was to investigate effect of standardized aqueous extract of flowering tops of Trifolium pratense L. on insulin resistance and SIRT1 expression in type 2 diabetic rats. Type 2 diabetes was induced by feeding high fat diet and administering low dose of streptozotocin. Diabetic animals were treated with standardized aqueous extract at three different doses. Parameters such as blood glucose, lipid profile, glycohemoglobin, insulin sensitivity, HOMA-IR and liver glycogen content were measured. Changes in morphology and expression of SIRT1 in pancreatic tissue were measured in histopathological and immunohistological studies. Aqueous extract treatment showed reduction in hyperglycemia and improved insulin sensitivity. Extract treatment also showed reduction in formation of glycated hemoglobin and improved liver glycogen level. Histopathological study revealed protecting effect of extract in pancreatic tissue against hyperglycemia induced damage. Treatment increased expression of SIRT1 in rat pancreatic tissue. Results indicate that the aqueous extract of Trifolium pratense had beneficial role in improving insulin sensitivity and SIRT1 expression.

Growth and elemental uptake of Trifolium repens in response to biochar addition, arbuscular mycorrhizal fungi and phosphorus fertilizer applications in low-Cd-polluted soils.

The aim of this study was to examine the effects of arbuscular mycorrhizal (AM) fungi, biochar (BC) addition and phosphorus (P) fertilizer applications on the mycorrhizal response, biomass and elemental uptake of Trifolium repens in cadmium (Cd)-polluted soils. The results showed that mycorrhizal colonization were significantly decreased by 100 mg P kg-1 fertilizer input. Moreover, AM fungi, BC addition and P fertilizer significantly increased shoot biomass accumulation at all treatments. In the absence of BC, the nitrogen (N), potassium (K), calcium (Ca) and magnesium (Mg) contents in the shoots were not affected by AM fungi after P fertilizer application, but the P content in the shoots significantly increased in response to AM fungi. In the absence of BC, both AM fungi and P fertilizer significantly reduced the Cd concentrations in the plant tissues as well as the soil diethylenetriaminepentaacetic acid (DTPA)-Cd concentration. These results indicated that the translocation factors (TFs) were influenced only by BC addition and that the roots could accumulate greater amounts of Cd than the shoots. On the basis of the hygienic standard for feed in China, the shoot Cd concentration in white clover was below the maximum permitted Cd concentration (1 µg g-1) across all treatments. Therefore, it is suggested that no negative mycorrhizal-white clover symbiotic relationships were observed and T. repens could be a suitable forage species for planting in soils with low concentrations of Cd contamination when BC and P fertilizer are applied.

Ensiled pulp from biorefining increased milk production in dairy cows compared with grass-clover silage.

The objective of the current study was to examine the effect of fibrous pulp and partial substitution of soybean meal with green protein concentrate from biorefining of grass-clover on dry matter intake, milk production, digestibility, and eating behavior in dairy cows compared with untreated grass-clover silage and soybean meal. Biorefining of grass-clover occurred right after harvest in a production-scale twin-screw press. The twin-screw pressing separated the grass-clover into a pulp and a green juice. The green juice was fermented using lactic acid bacteria for protein precipitation and then decanted, and the precipitate was heat dried to constitute the green protein concentrate. From the same field, grass-clover was harvested 6 d later due to rainy weather and was prewilted before ensiling. The pulp and the grass-clover were ensiled in bales without additives. The production trial consisted of an incomplete 6 × 4 Latin square trial (3-wk periods; 12 wk total) including 36 lactating Holstein cows. The trial had 6 treatments in a 2 × 3 factorial design with 2 forage types (grass-clover silage and pulp silage) and 3 protein treatments (low protein, high protein with soybean meal, and high protein with a mixture of soybean meal and green protein). The trial was designed to test silage type, protein type, protein level, and the interaction between protein level and silage type. The forage:concentrate ratio was 55:45 in low protein total mixed rations (TMR) and 51:49 in high protein TMR. Low protein and high protein TMR were composed of 372 and 342 g/kg of DM of experimental silages, respectively, and green protein supplemented TMR was composed of 28.5 g/kg of DM of green protein. Silage type did not affect dry matter intake of cows. The average energy-corrected milk yield was 37.0 and 33.4 kg/d for cows fed pulp silage and grass-clover silage, respectively, resulting in an improved feed efficiency in the cows receiving pulp silage. Milk fat concentration was greater in milk from cows fed pulp silage, and milk protein concentration was lower compared with milk from cows fed grass-clover silage. The in vivo digestibility of crude protein and neutral detergent fiber was greater for pulp silage diets compared with grass-clover silage diets. Eating rate was greater, whereas daily eating duration was lower, for pulp silage diets compared with grass-clover silage diets. The partial substitution of soybean meal with green protein did not affect dry matter intake, milk yield, or eating behavior. The in vivo digestibility of crude protein in green protein supplemented diets was lower compared with soybean meal diets. The results imply that extraction of protein from grassland plants can increase the value of the fiber part of grassland plants.

Globular structures in roots accumulate phosphorus to extremely high concentrations following phosphorus addition.

Crops with improved uptake of fertilizer phosphorus (P) would reduce P losses and confer environmental benefits. We examined how P-sufficient 6-week-old soil-grown Trifolium subterraneum plants, and 2-week-old seedlings in solution culture, accumulated P in roots after inorganic P (Pi) addition. In contrast to our expectation that vacuoles would accumulate excess P, after 7 days, X-ray microanalysis showed that vacuolar [P] remained low (<12 mmol kg-1 ). However, in the plants after P addition, some cortex cells contained globular structures extraordinarily rich in P (often >3,000 mmol kg-1 ), potassium, magnesium, and sodium. Similar structures were evident in seedlings, both before and after P addition, with their [P] increasing threefold after P addition. Nuclear magnetic resonance (NMR) spectroscopy showed seedling roots accumulated Pi following P addition, and transmission electron microscopy (TEM) revealed large plastids. For seedlings, we demonstrated that roots differentially expressed genes after P addition using RNAseq mapped to the T. subterraneum reference genome assembly and transcriptome profiles. Among the most up-regulated genes after 4 hr was TSub_g9430.t1, which is similar to plastid envelope Pi transporters (PHT4;1, PHT4;4): expression of vacuolar Pi-transporter homologs did not change. We suggest that subcellular P accumulation in globular structures, which may include plastids, aids cytosolic Pi homeostasis under high-P availability.

Effects of Heat Treatment of Green Protein on in Situ Protein Disappearance and in Vitro Fatty Acid Biohydrogenation.

Soluble protein extracted from leaves and stems of grasses and forage legumes is defined as green protein. The present study was conducted to evaluate in situ green protein degradability, intestinal protein disappearance, and in vitro fatty acids biohydrogenation (BH) in dairy cows. Three green protein concentrates (red clover, ryegrass, and grass clover) were heat treated as follows: oven-drying at 70 °C, subsequent autoclaving at 121 °C for 45 min, and for grass clover also spin flash-drying. Freeze-dried green protein was considered as a control (untreated). Autoclaving and oven-drying of green protein reduced the crude protein and dry matter degradability. The linolenic acid BH rate was lowest in heat-treated grass clover concentrate ( P < 0.01). In conclusion, green proteins are heat sensitive, and oven-drying can be an appropriate method to increase the amount of protein and unsaturated fatty acids escaping from the rumen.

Relationship between concentration of rare earth elements in soil and their distribution in plants growing near a frequented road.

Rare earth elements (REEs) are a group of elements whose concentration in numerous environmental matrices continues to increase; therefore, the use of biological methods for their removal from soil would seem to be a safe and reasonable approach. The aim of this study was to estimate the phytoextraction efficiency and distribution of light and heavy (LREEs and HREEs) rare earth elements by three herbaceous plant species: Artemisia vulgaris L., Taraxacum officinale F.H. Wigg. and Trifolium repens L., growing at a distance of 1, 10, and 25 m from the edge of a frequented road in Poland. The concentration of REEs in soil and plants was highly correlated (r > 0.9300), which indicates the high potential of the studied plant species to phytoextraction of these elements. The largest proportion of REEs was from the group of LREEs, whereas HREEs comprised only an inconsiderable portion of the REEs group. The dominant elements in the group of LREEs were Nd and Ce, while Er was dominant in the HREEs group. Differences in the amounts of these elements influenced the total concentration of LREEs, HREEs, and finally REEs and their quantities which decreased with distance from the road. According to the Friedman rank sum test, significant differences in REEs concentration, mainly between A. vulgaris L., and T. repens L. were observed for plants growing at all three distances from the road. The same relation between A. vulgaris L. and T. officinale was observed. The efficiency of LREEs and REEs phytoextraction in the whole biomass of plants growing at all distances from the road was A. vulgaris L. > T. officinale L. > T. repens L. For HREEs, the same relationship was recorded only for plants growing at the distance 1 m from the road. Bioconcentration factor (BCF) values for LREEs and HREEs were respectively higher and lower than 1 for all studied plant species regardless of the distance from the road. The studied herbaceous plant species were able to effectively phytoextract LREEs only (BCF > 1); therefore, these plants, which are commonly present near roads, could be a useful tool for removing this group of REEs from contaminated soil.

Effect of substituting fresh-cut perennial ryegrass with fresh-cut white clover on bovine milk fatty acid profile.

BACKGROUND: Including forage legumes in dairy systems can help address increasing environmental/economic concerns about perennial ryegrass monoculture pastures. This work investigated the effect of substituting fresh-cut grass with increasing quantities of fresh-cut white clover (WC) on milk fatty acid (FA) profile and transfer efficiency of dietary linoleic (LA) and α-linolenic (ALNA) acids to milk fat. Three groups of three crossbred dairy cows were used in a 3 × 3 crossover design. Dietary treatments were 0 g kg-1 WC + 600 g kg-1 grass, 200 g kg-1 WC + 400 g kg-1 grass, and 400 g kg-1 WC + 200 g kg-1 grass. All treatments were supplemented with 400 g kg-1 concentrates on a dry matter basis. Cows had a 19-day adaptation period to the experimental diet before a 6-day measurement period in individual tie stalls. RESULTS: Increasing dietary WC did not affect dry matter intake, milk yield or milk concentrations of fat, protein or lactose. Milk polyunsaturated FA concentrations (total n-3, total n-6, LA and ALNA) and transfer efficiency of LA and ALNA were increased with increasing dietary WC supply. CONCLUSION: Inclusion of WC in pastures may increase concentrations of nutritionally beneficial FA, without influencing milk yield and basic composition, but any implications on human health cannot be drawn. © 2018 The Authors. Journal of the Science of Food and Agriculture published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry.

Se enhanced phytoremediation of diesel in soil by Trifolium repens.

A pot-culture experiment was conducted to assess the effects of selenium (Se) (0.5 mg kg-1) on Trifolium repens exposed to various levels of diesel (0, 15, 20, 25 g kg-1) for 30 days and 60 days. Exposure to diesel for 60 day led to concentration-dependent decreases in root morphogenesis, chlorophyll content and CAT activity, and to dose-dependent increases in MDA content and SOD activity. The residual diesel concentration in soil increased and the removal efficiency decreased with soil diesel concentration. The chlorophyll content and residual diesel concentration after were slightly higher at 30 days than at 60days. Application of Se to soil increased Trifolium repens tolerance to diesel and significantly increased the phytoremediation effect at 60 days, with a removal rate of 36 ±â€¯8%, compared to 28 ±â€¯7% in the control. These results contribute to the ongoing effort to develop an effective phytoremediation system for soils highly contaminated by diesel.

Utilization of protein in red clover and alfalfa silages by lactating dairy cows and growing lambs.

Feeding trials were conducted with lactating cows and growing lambs to quantify effects of replacing dietary alfalfa silage (AS) with red clover silage (RCS) on nutrient utilization. The lactation trial had a 2 × 4 arrangement of treatments: AS or RCS fed with no supplement, rumen-protected Met (RPM), rumen-protected Lys (RPL), or RPM plus RPL. Grass silage was fed at 13% of dry matter (DM) with AS to equalize dietary neutral detergent fiber (NDF) and crude protein contents. All diets contained (DM basis) 5% corn silage and 16% crude protein. Thirty-two multiparous (4 ruminally cannulated) plus 16 primiparous Holstein cows were blocked by parity and days in milk and fed diets as total mixed rations in an incomplete 8 × 8 Latin square trial with four 28-d periods. Production data (over the last 14 d of each period) and digestibility and excretion data (at the end of each period) were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC). Although DM intake was 1.2 kg/d greater on AS than RCS, milk yield and body weight gain were not different. However, yields of fat and energy-corrected milk as well as milk content of fat, true protein, and solids-not-fat were greater on AS. Relative to AS, feeding RCS increased milk and energy-corrected milk yield per unit of DM intake, milk lactose content, and apparent N efficiency and reduced milk urea. Relative to AS, apparent digestibility of DM, organic matter, NDF, and acid detergent fiber were greater on RCS, whereas apparent and estimated true N digestibility were lower. Urinary N excretion and ruminal concentrations of ammonia, total AA, and branched-chain volatile fatty acids were reduced on RCS, indicating reduced ruminal protein degradation. Supplementation of RPM increased intake, milk true protein, and solids-not-fat content and tended to increase milk fat content. There were no silage × RPM interactions, suggesting that RPM was equally limiting on both AS and RCS. Supplementation of RPL did not influence any production trait; however, a significant silage × RPL interaction was detected for intake: RPL reduced intake of AS diets but increased intake of RCS diets. Duplicated metabolism trials were conducted with lambs confined to metabolism crates and fed only silage. After adaptation, collections of silage refusals and excreta were made during ad libitum feeding followed by feeding DM restricted to 2% of body weight. Intake of DM was not different when silages were fed ad libitum. Apparent digestibility of DM, organic matter, NDF, and hemicellulose was greater in lambs fed RCS on both ad libitum and restricted intake; however, acid detergent fiber digestibility was only greater at restricted intake. Apparent and estimated true N digestibility was substantially lower, and N retention was reduced, on RCS. Results confirmed greater DM and fiber digestibility in ruminants and N efficiency in cows fed RCS. Specific loss of Lys bioavailability on RCS was not observed. Based on milk composition, Met was the first-limiting AA on both silages; however, Met was not limiting based on production and nutrient efficiency. Depressed true N digestibility suggested impaired intestinal digestibility of rumen-undegraded protein from RCS.

Red Clover Aryl Hydrocarbon Receptor (AhR) and Estrogen Receptor (ER) Agonists Enhance Genotoxic Estrogen Metabolism.

Many women consider botanical dietary supplements (BDSs) as safe alternatives to hormone therapy for menopausal symptoms. However, the effect of BDSs on breast cancer risk is largely unknown. In the estrogen chemical carcinogenesis pathway, P450 1B1 metabolizes estrogens to 4-hydroxylated catechols, which are oxidized to genotoxic quinones that initiate and promote breast cancer. In contrast, P450 1A1 catalyzed 2-hydroxylation represents a detoxification pathway. The current study evaluated the effects of red clover, a popular BDS used for women's health, and its isoflavones, biochanin A (BA), formononetin (FN), genistein (GN), and daidzein (DZ), on estrogen metabolism. The methoxy estrogen metabolites (2-MeOE1, 4-MeOE1) were measured by LC-MS/MS, and CYP1A1 and CYP1B1 gene expression was analyzed by qPCR. Nonmalignant ER-negative breast epithelial cells (MCF-10A) and ER-positive breast cancer cells (MCF-7) were derived from normal breast epithelial tissue and ER+ breast cancer tissue. Red clover extract (RCE, 10 µg/mL) and isoflavones had no effect on estrogen metabolism in MCF-10A cells. However, in MCF-7 cells, RCE treatments downregulated CYP1A1 expression and enhanced genotoxic metabolism (4-MeOE1/CYP1B1 > 2-MeOE1/CYP1A1). Experiments with the isoflavones showed that the AhR agonists (BA, FN) preferentially induced CYP1B1 expression as well as 4-MeOE1. In contrast, the ER agonists (GN, DZ) downregulated CYP1A1 expression likely through an epigenetic mechanism. Finally, the ER antagonist ICI 182,780 potentiated isoflavone-induced XRE-luciferase reporter activity and reversed GN and DZ induced downregulation of CYP1A1 expression. Overall, these studies show that red clover and its isoflavones have differential effects on estrogen metabolism in "normal" vs breast cancer cells. In breast cancer cells, the AhR agonists stimulate genotoxic metabolism, and the ER agonists downregulate the detoxification pathway. These data may suggest that especially breast cancer patients should avoid red clover and isoflavone based BDSs when making choices for menopausal symptom relief.