Floral procyanidins of the forage legume red clover (Trifolium pratense L.)

The chemical characteristics of the purified procyanidin polymers of the flowers of the forage legume red clover (Trifolium pratense L.) were studied by (13)C NMR, acid-catalyzed degradation with benzyl mercaptan, and electrospray ionization mass spectrometry (ESI-MS). The (13)C NMR showed that the fraction consisted of predominantly procyanidin polymers. The thiolysis reaction products indicated a mean degree of polymerization (mDP) of 9.3 with epicatechin (81%) as the abundant flavan-3-ol extension unit and the terminating units dominated by catechin (95%). ESI-MS showed a range of oligomeric procyanidin ions (DP of 2-11). The white clover floral prodelphinidins consist of terminal units with nearly equal proportions of epigallocatechin (52%) and gallocatechin (48%) and extender units showing epigallocatechin (56%) and gallocatechin (39%). The dramatic difference in the stereochemistry of the terminal and extender units observed for the red clover floral procyanidins contrasts with the mixture of cis and trans stereochemistry observed for white clover floral prodelphinidins.     

Comparison of the in vitro estrogenic activities of compounds from hops (Humulus lupulus) and red clover (Trifolium pratense)

Because the prevailing form of hormone replacement therapy is associated with the development of cancer in breast and endometrial tissues, alternatives are needed for the management of menopausal symptoms. Formulations of Trifolium pratense L. (red clover) are being used to alleviate menopause-associated hot flashes but have shown mixed results in clinical trials. The strobiles of Humulus lupulusL. (hops) have been reported to contain the prenylflavanone, 8-prenylnaringenin (8-PN), as the most estrogenic constituent, and this was confirmed using an estrogen receptor ligand screening assay utilizing ultrafiltration mass spectrometry. Extracts of hops and red clover and their individual constituents including 8-PN, 6-prenylnaringenin (6-PN), isoxanthohumol (IX), and xanthohumol (XN) from hops and daidzein, formononetin, biochanin A, and genistein from red clover were compared using a variety of in vitro estrogenic assays. The IC50 values for the estrogen receptor alpha and beta binding assays were 15 and 27 microg/mL, respectively, for hops and 18.0 and 2.0 microg/mL, respectively, for the red clover extract. Both of the extracts, genistein, and 8-PN activated the estrogen response element (ERE) in Ishikawa cells while the extracts, biochanin A, genistein, and 8-PN, significantly induced ERE-luciferase expression in MCF-7 cells. Hop and red clover extracts as well as 8-PN up-regulated progesterone receptor (PR) mRNA in the Ishikawa cell line. In the MCF-7 cell line, PR mRNA was significantly up-regulated by the extracts, biochanin A, genistein, 8-PN, and IX. The two extracts had EC50 values of 1.1 and 1.9 microg/mL, respectively, in the alkaline phosphatase induction assay. On the basis of these data, hops and red clover could be attractive for the development as herbal dietary supplements to alleviate menopause-associated symptoms.     

Effect of dinoseb on nitrogen fixation of red clover (Trifolium pratense)

The effect of the herbicide dinoseb (2-sec-butyl-4,6-dinitrophenol) on the symbiotic nitrogen fixation of red clover (Trifolium pratense cv. Venla) was studied in field, greenhouse and laboratory experiments. In the field, the herbicide reduced the levels of nitrogenase (C₂H₂) activity of the plants up to 18 days after treatment, but increased the yields compared to hand-weeded and unweeded controls. Rhizobium strains isolated from root nodules of plants treated with dinoseb showed resistance to the herbicide when grown on laboratory media containing dinoseb. However, in a pot experiment, inoculation with a strain which grew in the presence of 200 μg dinoseb ml^?1 did not improve the performance of red clover treated with dinoseb. In the pot experiment dinoseb inhibited nitrogenase activity when sprayed on the leaves, but not when added to the growth medium. Application of starter nitrogen did not influence the effect of dinoseb. The results suggest that dinoseb did not act directly on the nodules, but affected nitrogen fixation by damaging the photosynthetic parts of the plant.     

LC-ESI-MS study of the flavonoid glycoside malonates of red clover (Trifolium pratense)

High-performance liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) was applied to the analysis of the flavonoids and their glycoside malonates of the flowers and leaves of red clover (Trifolium pratense). Through LC-MS comparative studies on the plant extracts and their malonate-free extracts, approximately 20 flavonoid glycoside malonates were detected in the flower extract. Eight were identified as genistin 6' '-O-malonate (39), formononetin 7-O-beta-D-glucoside 6' '-O-malonate (40), biochanin A 7-O-beta-D-glucoside 6' '-O-malonate (41), trifoside 6' '-O-malonate (42), irilone 4'-O-beta-D-glucoside 6' '-O-malonate (43), pratensein 7-O-beta-D-glucoside 6' '-O-malonate (44), isoquercitrin 6' '-O-malonate (45), and 3-methylquercetin 7-O-beta-D-glucoside 6' '-O-malonate (46). About 15 other flavonoids and clovamides were proved to be present in this extract. The study also found that the flowers contained flavones as the major flavonoids, whereas the leaves had isoflavones as the major flavonoids. This is the first detection of the six malonates (39 and 42-46) in the extracts of red clover, and among them, 42, 43, and 46 are new compounds.     

Development of nitrification hot-spots around degrading red clover (Trifolium pratense) leaves in soil

 Litterbags with clover leaves in soil were buried in the field to study the influence of degrading clover leaves on the development of elevated nitrification activity (hot-spots) in space and time. Potential NH₃ oxidation activity indicating the population size of ammonia-oxidizing bacteria was measured in soil samples taken in a zone a few millimetres thick around the leaves and in bulk soil. On 3 sampling days during leaf degradation, the potential NH₄ ⁺ oxidation was significantly higher in leaf-associated soil than in bulk soil, the largest difference (factor of 2) occurring 24 days after burial. At all sampling occasions, NH₄ ⁺ oxidation rates followed a normal distribution, except for a log-normal distribution in the leaf-associated soil sampled 24 days after burial when some very active samples (hot-spots) appeared. In a similar laboratory experiment we examined the effects of soil water on the development of nitrification hot-spots. We observed that the development of hot-spots did not take place in soil incubated at 60% of water-holding capacity (WHC), whereas they developed faster when the soil was incubated at approximately 100% WHC. It was concluded that soil water in combination with easily degradable organic N were essential parameters for the development of nitrification hot-spots in this soil. Received: 18 April 2000     

Specific polyphenol oxidase activity of red clover (Trifolium pratense) and its relation with forage quality in field experiments

Abstract

In feeding studies, red clover (RC) influenced positively the N utilization by ruminants. A relationship between polyphenol oxidase (PPO) activity and forage quality has not been established. Our objective was to investigate seasonal, site, genotype, and management effects on specific PPO activity in RC in three experiments under field conditions, and relate the activity to forage-quality parameters. In Experiment 1, six RC genotypes at two study sites were submitted to a 3-cut system. Specific PPO activity, forage quality, and vegetation stage were determined. PPO activity varied between harvest and study sites, with genotypes differing up to 3-fold in PPO activity within harvests. The specific PPO activity, forage quality, and vegetation stage in RC subjected to 5-cut system and grazing (Experiment 2) were determined. Additionally, in Experiment 3, cutting frequency in RC swards including mechanical stress (rolling) was investigated. The induction of PPO activity in RC by grazing or mechanical stress (Experiments 2 and 3) increased the activity up to 2.5-fold compared with RC at similar vegetation stage submitted to the 5-cut system. Mechanical stress induced by grazing or rolling, and seasonal differences, seem to have a larger influence on specific PPO activity than does the genotype effect observed in Experiment 1. For forage quality, an increased specific PPO activity explained 29–46% of the reduction in protein fraction ‘A’ content (non-protein N) in the cutting systems in Experiments 2 and 3. Other CP fractions achieved a lower relation. Furthermore, the precipitation-to-temperature ratio preceeding a harvest explained 63% of the variation in the specific PPO activity. In conclusion, the PPO activity in RC is induced by grazing and rolling. Whereas weather conditions preceeding a harvest showed a large influence, genotype influence had only minor relevance. These results may have implications for regional harvest management towards efficient N utilization by ruminants.     

Three polyphenol oxidases from red clover (Trifolium pratense) differ in enzymatic activities and activation properties

Polyphenol oxidases (PPOs) oxidize o-diphenols to o-quinones, which cause browning reactions in many wounded fruits, vegetables, and plants including the forage crop red clover (Trifolium pratense L.). Production of o-quinones in red clover inhibits postharvest proteolysis during the ensiling process. The cDNAs encoding three red clover PPOs were expressed individually in alfalfa (Medicago sativa L.), which lacks detectable endogenous foliar PPO activity and o-diphenols. Several physical and biochemical characteristics of the red clover PPOs in alfalfa extracts were determined. In transgenic alfalfa extracts, red clover PPOs exist in a latent state and are activated (10-40-fold increase in activity) by long incubations (>2 days) at ambient temperature or short incubations (<10 min) at > or =65 degrees C. PPO1 appears to be more stable at high temperatures than PPO2 or PPO3. During incubation at ambient temperature, the molecular masses of the PPO enzymes were reduced by approximately 20 kDa. The apparent pH optima of latent PPO1, PPO2, and PPO3 are 5.5, 6.9, and 5.1, respectively, and latent PPO1 is slightly activated (~5-fold) by low pH. Activation of the PPOs shifts the pH optima to approximately 7, and the activated PPOs retain substantial levels of activity as the pH increases above their optima. The latent and activated PPOs were surveyed for ability to oxidize various o-diphenols, and activation of the PPOs had little effect on substrate specificity. Activation increases the V max but not the affinity of the PPO enzymes for caffeic acid. Results indicate red clover PPOs undergo structural and kinetic changes during activation and provide new insights to their effects in postharvest physiology.     

Potassium uptake of rye-grass (Lolium perenne) and red clover (Trifolium pratense) as related to root parameters

Summary Rye-grass (Lolium perenne) is known to be a strong competitor to red clover (Trifolium pratense) for soil K⁺ under conditions of low K availability in the soil. The objective of this study was to clarify whether this competitive behaviour of the two species can be explained by root morphology. Total K⁺ uptake ofL. perenne andT. pratense was studied under field conditions in relation to root fresh weight, root density, root cation exchange capacity, root surface and root length. The soil was an Alfisol, Udalf. All root parameters, when calculated per unit soil surface (M²), were higher inL. perenne than inT. pratense. In addition,L. perenne had longer root hairs and a denser root hair system thanT. pratense. The greatest difference in root morphology between species was root length, withL. perenne roots averaging 4–6 times longer than those ofT. pratense.Significant correlations were found between the total K⁺ uptake and all root parameters examined, with highest correlationsforroot fresh weight (r,0.92***T. pratense; 0.94***L. perenne) and root length (r, 0.91***T. pratense;r, 0.93***L. perenne). Potassium uptake per unit root fresh weight, root surface and root length were all significantly higher forT. pratense than for L. perenne. Differences in the rate of K⁺ uptake between species were particularly high when expressed per unit root length. Because of its greater root length and surface area,L. perenne can take up more soil K⁺ thanT. pratense, particularly where there is a low K^– supply in the soil. Under such conditionsL. perenne will be a particularly strong competitor toT. pratense.     

Inhibition of extrahepatic human cytochromes P450 1A1 and 1B1 by metabolism of isoflavones found in Trifolium pratense (red clover)

Biochanin A and formononetin are the predominant isoflavones in red clover. In a previous study (J. Agric. Food Chem. 2002, 50, 4783-4790), it was demonstrated that human liver microsomes converted biochanin A and formononetin to genistein and daidzein. This paper now shows CYP1B1-catalyzed O-demethylation of biochanin A and formononetin to produce genistein and daidzein, respectively, which inhibit CYP1B1. Recombinant human CYP1A1 or CYP1B1 was incubated with biochanin A or formononetin. CYP1A1 catalyzed isoflavone 4'-O-demethylation and hydroxylations with similar efficiency, whereas CYP1B1 favored 4'-O-demethylation over hydroxylations. Three of the biochanin A metabolites (5,7,3'-trihydroxy-4'-methoxyisoflavone, 5,7,8-trihydroxy-4'-methoxyisoflavone, and 5,6,7-trihydroxy-4'-methoxyisoflavone) were characterized by 1H NMR spectroscopy and mass spectrometry. Daidzein (Ki = 3.7 microM) exhibited competitive inhibition of CYP1B1 7-ethoxyresorufin O-deethylase activity, and genistein (Ki = 1.9 microM) exhibited mixed inhibition. Biochanin A and/or formononetin may exert anticarcinogenic effects directly by acting as competitive substrates for CYP1B1 or indirectly through their metabolites daidzein and genistein, which inhibit CYP1B1.     

Effect of sowing methods and sowing rate in organic seed production of timothy (Phleum pratense L.), meadow fescue (Festuca pratensis Huds.) and red clover (Trifolium pratense L.)

Different sowing methods and sowing rates were evaluated in organic seed production of timothy (two trials), meadow fescue (two trials) and red clover (one trial) in Southeast Norway, during 2010–2013. The plan included: (1) broadcast sowing of grass/clover, cover crop sown at 12 cm row distance; (2) sowing of cover and seed crop in crossed rows, both at 12 cm row distance; and (3) sowing of cover crop and seed crop in every other row. The three sowing rates were 5, 10 and 15 kg ha–1 in timothy and meadow fescue and 3, 6 and 9 kg ha–1 in red clover. On average for sowing rates and all trials with timothy, meadow fescue and red clover, first year’s seed yields were 5–6%, 20–25% and 19–25% higher on plots sown with cover crop and seed crop in every other row than on plots where seed crop had been broadcast or sown perpendicularly to the cover crop. The different sowing methods had no effect on weed coverage or weed contamination in the cleaned seed. Increasing sowing rate usually had a negative influence on seed yield, while weed coverage/contamination was not significantly affected. It is concluded that organic seed crops should be established with cover crop and seed crop in every other row at a low sowing rate. However, in an organic production system, even this favorable method will not always be sufficient to meet the requirement for seed crop purity.     

Kinetics of inorganic and organic P release from red clover (Trifolium pratense L.) and ryegrass (Lolium multiflorum L.) upon frost or drying

An incubation study with fresh, frozen, and dried clover and rye-grass shoots gathered in autumn was performed in the laboratory (43 days). The aim was to study inorganic and organic phosphorus (P) release from plant material during decomposition without soil. Plant materials (2?cm size) were mixed with small glass beads and placed in large syringes for consecutive water extractions on 7 occasions. Leachates were analysed for inorganic and total P. At the first leaching event, 8% of total crop P was released from fresh, 24% from frozen and 27% from dried plant material. During decomposition, both inorganic and organic P was released following first order kinetics, with grass releasing mostly inorganic P and clover mostly organic P. After 43 days, 42%–50% of total crop P from fresh, 49%–51% from frozen and 57%–69% from dried material was released, with significant differences between treatments. Using the results in calculations on field scale showed that P released from overwintering crops under cold climate conditions can amount to several kg P ha^?1. Thus, senescence of overwintering aboveground biomass can be a significant source for P leaching from soils.     

Leaf size variation in subterranean clover (Trifolium subterraneum L. sensu lato)

In subterranean clover, leaf size contributes to plant competitivity; besides, this trait is attributed a diagnostic relevance. The study assessed leaf size variation at two growth stages (60 days after sowing and flowering date) on genotypes belonging to the three main subspecies of the complex, viz. subsp. subterraneum, brachycalycinum, and yanninicum. After 60 days, brachycalycinum showed the largest leaflet area while subterraneum and yanninicum did not differ. Leaf size increase between the two measurements, remarkable in all subspecies, was significantly highest in yanninicum, which showed the largest area at flowering date. The leaflet length/breadth ratio was similar in the three subspecies at both stages. In subterraneum and brachycalycinum this ratio decreased significantly from the first to the second measurement; leaflet area increase seemed depending more on breadth than length increase. Yanninicum showed the longest petiole at flowering, and subspecies ranking for this character paralleled that of leaflet area. Particularly in subterraneum, there was a noticeable genotype variation for all characters. Earlier genotypes tended to have larger leaves after 60 days, and shorter petioles and smaller leaves at flowering date. In subterraneum, the genotype leaflet size decreased on increasing altitude and rainfall, and decreasing temperature of the collection sites. The leaf characters examined fairly confirmed to bear a taxonomic basis. Further assessing the physiological behaviour, particularly the response to defoliation and its agronomic implications, of genotypes characterised by different leaf size is envisageable.     

Repellency of insecticides and the effect of thiacloprid on bumble bee colony development in red clover (Trifolium pratense L.) seed crops

The study intended to compare repellency of three insecticides on bumble bees and honey bees in Norwegian red clover (Trifolium pratense L.) seed crops, and to examine effects of thiacloprid on bumble bee colony development in the field. The repellency study was carried out in a large-scale field trial in SE Norway in 2013. On average for observations during the first week after spraying, 17 and 40% less honey bees (P?=?.03) and 26 and 20% less bumble bees (P?=?.36) were observed on plots sprayed with the pyrethroids lambda-cyhalothrin and alpha-cypermethrin, respectively, than on unsprayed control plots. No pollinator repellency was found on plots sprayed with the neonicotinoid thiacloprid. Compared with unsprayed control the seed yield increases were 22% on plots sprayed with thiacloprid vs. 12–13% on plots sprayed with pyrethroids (P?=?.10). Follow-up studies in 2014–2016 focused on the effect of thiacloprid on bumble bee colony development in commercially reared nests of Bombus terrestris placed into red clover seed crops at the start of flowering. Unsprayed control crops were compared with crops sprayed either at the bud stage or when 18–44% of flower heads were in full bloom. Chemical analyses of adult bumble bees showed that thiacloprid was taken up in bees when crops were sprayed during flowering, but not detected when crops were sprayed at the bud stage. The bumble bees in late-sprayed crops also developed weaker colonies than in unsprayed crops. Dead bees with a high internal concentration of thiacloprid were found in one crop sprayed during the night at 35% flowering. This shows that thiacloprid is not bee-safe if sprayed after anthesis and that spraying has to be conducted at the bud stage to reduce its contamination of nectar and pollen.     

Measurement of pH at the root surface of red clover (Trifolium pratense) grown in soils differing in proton buffer capacity

Summary Measurements of pH were made at the root surface of Trifolium pratense, using Sb electrodes. Nodulated plants were grown in rhizotrones on a sandy soil free of carbonate and on a clay soil rich in carbonate. In the sandy soil, pH at the surface of root laterals was about 1 unit lower than in the bulk soil. The lowest pH values were found at the root tips. In the calcareous soil, pH measured at the root surface did not differ from pH in the bulk soil. This soil had a much higher H⁺ buffer capacity than the sandy soils. It seems likely that H⁺ ions excreted from the roots grown in the calcareous soil were directly neutralized by soil carbonate.     

Extraction of isoflavone malonylglucosides from Trifolium pratense L

Extraction of isoflavone malonylglucosides from red clover (Trifolium pratense L.) is a complicated procedure. This is due to the relatively unstable character of the thermolabile glucoside malonates as well as by action of native beta-glucosidases, resulting in a rapid degradation of malonylated glucosides into their corresponding aglucones. In this study, Tris was identified as a suitable beta-glucosidase inhibitor in red clover extracts, optimized at 350 mM Tris in 80% ethanol at pH 7.2. Extraction of fresh red clover leaves using Tris increased the concentration of malonate conjugated isoflavones approximately 13 to 24 times as opposed to extraction without Tris. A comparison of isoflavone profiles obtained after extraction with and without Tris of different plant organs of red clover and several species within the family Fabaceae suggests that the amount and/or activity of the degenerative beta-glucosidase enzymes vary for the different plant parts of red clover and among the species studied. Therefore, the use of standard extraction methods may well result in overestimation of the concentration of aglucones and consequently underestimation of the malonylglucoside isoflavones concentration depending on the plant species and plant part studied.     

Latent and active polyphenol oxidase (PPO) in red clover (Trifolium pratense) and use of a low PPO mutant to study the role of PPO in proteolysis reduction

Polyphenol oxidase (PPO) activity in leaf extracts of wild type (WT) red clover and a mutant line expressing greatly reduced levels of PPO (LP red clover) has been characterized. Both latent and active forms of PPO were present, with the latent being the predominant form. PPO enzyme and substrate (phaselic acid) levels fluctuated over a growing season and were not correlated. Protease activation of latent PPO was demonstrated; however, the rate was too low to have an immediate effect following extraction. A novel, more rapid PPO activation mechanism by the enzyme's own substrate was identified. Rates of protein breakdown and amino acid release were significantly higher in LP red clover extracts compared with WT extracts, with 20 versus 6% breakdown of total protein and 1.9 versus 0.4 mg/g FW of free amino acids released over 24 h, respectively. Inclusion of ascorbic acid increased the extent of protein breakdown. Free phenol content decreased during a 24 h incubation of WT red clover extracts, whereas protein-bound phenol increased and high molecular weight protein species were formed. Inhibition of proteolysis occurred during wilting and ensilage of WT compared with LP forage (1.9 vs 5 and 17 vs 21 g/kg of DM free amino acids for 24 h wilted forage and 90 day silage, respectively). This study shows that whereas constitutive red clover PPO occurs predominantly in the latent form, this fraction can contribute to reducing protein breakdown in crude extracts and during ensilage.     

Enzyme polymorphism and adaptation in strawberry clover (Trifolium fragiferum L.)

Enzymatic survey of eight loci in 22 populations of Trifolium fragiferum L. sampled in three regions in Poland showed regional differences with regard to five genetic parameters describing genetic polymorphism and variability. Differences were attributed to the following climatic variables: annual precipitation, temperature and solar radiation. Continental climatic conditions were favorable for accumulation of genetic polymorphism and variability. Vegetative reproduction versus sexual reproduction had a higher incidence rate under milder climatic conditions. This former mode of reproduction also affected patterns of distribution of variation within regions, by reducing the intra-populational component of total variability.     

Interaction Effects of Nitrogen and Phosphorus on Nodulation in Red Clover (Trifolium pratense L.)

Red clover (Trifolium pratense L.) is one of the most important plants in forage production, especially in northern areas. Fertilisation practices are focused on high yield and forage quality but effects of nutrients on nodulation and N₂ fixation are poorly understood. The aim of this work was to study how nitrogen (N) and phosphorus (P) separately as well as in combination affected nodulation. Red clover plants were grown in pots with gravel in a greenhouse for 11 weeks. To resemble field conditions the root temperature was kept lower than the shoot temperature. Plants were given five different combinations of N and P concentrations during growth. The result showed that at high N concentrations P had a counteracting effect on the N inhibition. The N₂-fixation parameters, nodule number, nodule dry matter and specific nitrogenase activity, were six times higher in plants grown with high N and high P than in plants with high N and low P. When the N₂-fixation parameters and the dry matter of roots and shoots were related to total plant dry matter, there was a stronger effect of P on nodulation parameters than on roots and shoots. This indicates that P has a direct effect on the N₂-fixation parameters, rather than an indirect effect via increased plant growth. These results demonstrate the importance to studying the effects of more than one nutrient at a time.     

Red clover Trifolium pratense L. phytoestrogens: UV-B radiation increases isoflavone yield, and postharvest drying methods change the glucoside conjugate profiles

Isoflavone extracts of red clover Trifolium pratense L. (cv. Pawera) with dissimilar glucoside conjugate profiles were obtained by employing different postharvest drying methods. The most prominent isoflavones found were formononetin and biochanin A and their corresponding glucosides and malonyl glucoside esters. Postharvest freeze drying inhibited the conversion of the glycosides to the aglycones, while vacuum drying allowed for maximum conversion of the glycosides to their corresponding aglycones. Air drying produced a low level of the aglycones formononetin and biochanin A, and oven drying promoted decarboxylation of the malonyl glucosides to the acetyl glucosides. Exposure to enhanced UV-B radiation resulted in an increase in total formononetin and biochanin A isoflavone levels, indicating that harvest during a period of high ambient UV-B radiation may be appropriate for maximum yield. The levels of caffeic acid and flavonols also increased by about 40 and 250%, respectively, on exposure to enhanced UV-B radiation.