Seasonal variation of red clover (Trifolium pratense L., Fabaceae) isoflavones and estrogenic activity

Red clover (Trifolium pratense L., Fabaceae) dietary supplements are currently used to treat menopausal symptoms because of their high content of the mildly estrogenic isoflavones daidzein, genistein, formononetin, and biochanin A. These compounds are estrogenic in vitro and in vivo, but little information exists on the best time to harvest red clover fields to maximize content of the isoflavones and thus make an optimal product. Samples of cultivated red clover above-ground parts and flower heads were collected in parallel over one growing season in northeastern Illinois. Generally, autohydrolytic extracts of above-ground parts contained more isoflavones and had more estrogenic activity in Ishikawa endometrial cells as compared with extracts of flower heads. Daidzein and genistein contents peaked around June to July, while formononetin and biochanin A contents peaked in early September. Flower head and total above-ground parts extracts exhibited differential estrogenic activity in an Ishikawa (endometrial) cell-based alkaline phosphatase induction assay, whereas nondifferential activity was observed for most extracts tested in an MCF-7 (breast) cell proliferation assay when tested at the same final concentrations. Ishikawa assay results could be mapped onto the extracts' content of individual isoflavones, but MCF-7 results did not show such a pattern. These results suggest that significant metabolism of isoflavones may occur in MCF-7 cells but not in Ishikawa cells; therefore, caution is advised in the choice of bioassay used for the biological standardization of botanical dietary supplements.     

Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptoms

Eight botanical preparations that are commonly used for the treatment of menopausal symptoms were tested for estrogenic activity. Methanol extracts of red clover (Trifolium pratense L.), chasteberry (Vitex agnus-castus L.), and hops (Humulus lupulus L.) showed significant competitive binding to estrogen receptors alpha (ER alpha) and beta (ER beta). With cultured Ishikawa (endometrial) cells, red clover and hops exhibited estrogenic activity as indicated by induction of alkaline phosphatase (AP) activity and up-regulation of progesterone receptor (PR) mRNA. Chasteberry also stimulated PR expression, but no induction of AP activity was observed. In S30 breast cancer cells, pS2 (presenelin-2), another estrogen-inducible gene, was up-regulated in the presence of red clover, hops, and chasteberry. Interestingly, extracts of Asian ginseng (Panax ginseng C.A. Meyer) and North American ginseng (Panax quinquefolius L.) induced pS2 mRNA expression in S30 cells, but no significant ER binding affinity, AP induction, or PR expression was noted in Ishikawa cells. Dong quai [Angelica sinensis (Oliv.) Diels] and licorice (Glycyrrhiza glabra L.) showed only weak ER binding and PR and pS2 mRNA induction. Black cohosh [Cimicifuga racemosa (L.) Nutt.] showed no activity in any of the above in vitro assays. Bioassay-guided isolation utilizing ER competitive binding as a monitor and screening using ultrafiltration LC-MS revealed that genistein was the most active component of red clover. Consistent with this observation, genistein was found to be the most effective of four red clover isoflavones tested in the above in vitro assays. Therefore, estrogenic components of plant extracts can be identified using assays for estrogenic activity along with screening and identification of the active components using ultrafiltration LC-MS. These data suggest a potential use for some dietary supplements, ingested by human beings, in the treatment of menopausal symptoms.     

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.     

红壤中重金属的复合污染及污染指数

A field experiment was conducted to study the effects of Pb,Cd,Cu,Zn and As coexisting in red soil on growth of rice (Oryza sativa L.) mung bean(Vigna rabiata(Linn.) Wilczek),alfalfa(Medicago sativa L.),Slash pine(Pinus elliottii Engelm.)and aspen(Populus L.).Results showed that rice,mung bean and alfalfa were significantly influenced by combined poollution of the heavy metals,the contents of Pb,Cd and As in rice grains greatly exceeded the National Standards for Food Hygiene of China.Heavy metals at a high concentration seriously retarded growth of mung bean and alfalfa,but not so obviously with slash pine and aspen.The composite index is suggested for evaluating the relativity of combined pollution with heavy metals in soil.     

Urolithins, ellagic acid-derived metabolites produced by human colonic microflora, exhibit estrogenic and antiestrogenic activities

Urolithins A and B (hydroxy-6H-dibenzo[b,d]pyran-6-one derivatives) are colonic microflora metabolites recently proposed as biomarkers of human exposure to dietary ellagic acid derivatives. Molecular models suggest that urolithins could display estrogenic and/or antiestrogenic activity. To this purpose, both urolithins and other known phytoestrogens (genistein, daidzein, resveratrol, and enterolactone) were assayed to evaluate the capacity to induce cell proliferation on the estrogen-sensitive human breast cancer MCF-7 cells as well as the ability to bind to alpha- and beta-estrogen receptors. Both urolithins A and B showed estrogenic activity in a dose-dependent manner even at high concentrations (40 microM), without antiproliferative or toxic effects, whereas the other phytoestrogens inhibited cell proliferation at high concentrations. Overall, urolithins showed weaker estrogenic activity than the other phytoestrogens. However, both urolithins displayed slightly higher antiestrogenic activity (antagonized the growth promotion effect of 17-beta-estradiol in a dose-dependent manner) than the other phytoestrogens. The IC(50) values for the ERalpha and ERbeta binding assays were 0.4 and 0.75 microM for urolithin A; 20 and 11 microM for urolithin B; 3 and 0.02 for genistein; and 2.3 and 1 for daidzein, respectively; no binding was detected for resveratrol and enterolactone. Urolithins A and B entered into MCF-7 cells and were metabolized to yield mainly urolithin-sulfate derivatives. These results, together with previous studies regarding absorption and metabolism of dietary ellagitannins and ellagic acid in humans, suggest that the gut microflora metabolites urolithins are potential endocrine-disrupting molecules, which could resemble other described "enterophytoestrogens" (microflora-derived metabolites with estrogenic/antiestrogenic activity). Further research is warranted to evaluate the possible role of ellagitannins and ellagic acid as dietary "pro-phytoestrogens".     

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.     

Evaluation of the phytoestrogenic activity of Cyclopia genistoides (honeybush) methanol extracts and relevant polyphenols

Unfermented C. genistoides methanol extracts of different harvestings and selected polyphenols were evaluated for phytoestrogenic activity by comparing binding to both ER subtypes, transactivation of an ERE-containing promoter reporter, proliferation of MCF-7-BUS and MDA-MB-231 breast cancer cells, and binding to SHBG. The extracts from one harvesting of C. genistoides (P104) bound to both ER subtypes. All extracts transactivated ERE-containing promoter reporters via ERbeta but not via ERalpha. All extracts, except P122, caused proliferation of the estrogen-sensitive MCF-7-BUS cells. Proliferation of MCF-7-BUS cells was ER-dependent as ICI 182,780 reversed proliferation. Physiologically more relevant, extracts antagonized E2-induced MCF-7-BUS cell proliferation. Furthermore, all extracts, except P122, induced proliferation of the estrogen-insensitive MDA-MB-231 cells, suggesting that the extracts are able to induce ER-dependent and ER-independent cell proliferation. Binding to SHBG by extracts was also demonstrated. These results clearly show that C. genistoides methanol extracts display phytoestrogenic activity and act predominantly via ERbeta. HPLC and LC-MS analysis, however, suggests that the observed phytoestrogenic activity cannot be ascribed to polyphenols known to be present in other Cyclopia species.     

Evaluation of estrogenic/antiestrogenic activity of ellagic acid via the estrogen receptor subtypes ERalpha and ERbeta

Ellagic acid is a plant-derived polyphenol, possessing antioxidant, antiproliferative, and antiatherogenic properties. Whether this compound has estrogenic/antiestrogenic activity, however, remains largely unknown. To answer this question, we first investigated the ability of ellagic acid to influence the activity of the estrogen receptor subtypes ERalpha and ERbeta in HeLa cells. Cells co-transfected with an estrogen response element (ERE)-driven luciferase (Luc) reporter gene and an ERalpha- or ERbeta-expression vector were exposed to graded concentrations of ellagic acid. At low concentrations (10(-7) to 10(-9) M), this compound displayed a small but significant estrogenic activity via ERalpha, whereas it was a complete estrogen antagonist via ERbeta. Further evaluation revealed that ellagic acid was a potent antiestrogen in MCF-7 breast cancer-derived cells, increasing, like the pure estrogen antagonist ICI182780, IGFBP-3 levels. Moreover, ellagic acid induced nodule mineralization in an osteoblastic cell line (KS483), an effect that was abolished by the estrogen antagonist. Endometrium-derived epithelial cells (Ishikawa) showed no response to the natural compound by using a cell viability assay (MTT). These findings suggest that ellagic acid may be a natural selective estrogen receptor modulator (SERM).     

Inoculation responses of perennial forage legumes grown in fresh hill‐land ultisols as affected by soil acidity‐related factors

A growth chamber experiment was initiated with two field moist, marginal and acidic (pH 5.1–5.2) soils of the Lily series (Typic Hapludults) in order to determine the need for improved legume‐rhizobia symbioses for forage species of current, or potential, use in the renovation of Appalachian hill‐land pastures. One soil was from an abandoned pasture having broomsedge (Andropogon virginicus L.) as the predominant vegetation, whereas the other was from a minimally‐managed pasture dominated by orchardgrass (Dactylis glomerata L.). Treatments included inoculation (or no inoculation) and the addition of aluminum, nil, or lime to provide a range of soil acidities. Both soils contained effective populations of naturalized rhizobia for white clover (Trifolium repens L.) and red clover (Trifolium pratense L.), but low and/or ineffective naturalized populations of rhizobia for alfalfa (Medicago sativa L.), birdsfoot trefoil (Lotus corniculatus L.), bigflower vetch (Vicia grandiflora Scop.), and flatpea (Lathyrus sylvestris L.). Seed inoculation, by lime‐pelleting, was highly beneficial in establishing effective symbioses for all these latter species. The addition of low levels of aluminum or lime (1.5 and 2.0 cmol/kg soil, respectively) had little effect on any of the symbioses, with the exception of those for alfalfa. Thus, an improved legume rhizobia symbiosis would not seem to be a prerequisite for renovating pastures established on chemically similar ultisols with the forage legume species examined in this study, especially if the pasture has at least some history of management.     

Forage yield and crude protein of interseeded legume‐bermudagrass mixtures as affected by phosphorus fertilizer 1

Bermudagrass (Cynodon dactylon L.) is a warm season perennial that is well adapted in the southern Great Plains. It is one of the region's most important forage crops used for livestock production, and is commonly grown without legume interseeding. Recent research has investigated ways of improving the quality and quantity of this forage. The objectives of this study were to determine the effect of interseeded legumes and phosphorus (P) fertilizer on bermudagrass pasture forage yield and crude protein content. One experiment was initiated in 1993 in eastern Oklahoma in an established bermudagrass pasture. Red clover (Trifolium pratense L.), ladino clover (Trifolium repens L.), and two varieties of alfalfa (Medicago sativah), ’alfagraze’ and'common’, were interseeded by hand into an established stand of bermudagrass. The effect of P on forage yield and crude protein was evaluated using a 30‐kg P ha^‐1 rate applied at establishment versus no applied P. Forage yield was collected three times throughout the growing season each year from 1994 through 1997. When both alfalfa varieties were interseeded into a bermudagrass pasture without applying additional P fertilizer, forage yields for the legume‐grass mixtures decreased below those obtained from the monoculture bermudagrass in the first year of the stand. The alfalfa variety ‘alfagraze’ interseeded into established bermudagrass decreased total forage yield over the entire 4‐yr study. Interseeded red clover and ladino clover increased crude protein of the forage compared with monoculture bermudagrass the first two years of the study, with red clover continuing to increase crude protein in the fourth year. However, when 30 kg P ha^‐1 was applied to the bermudagrass prior to establishment of the legumes, no change in yield or protein was observed for both alfalfa varieties’ interseeding treatments versus the unfertilized mixtures. Although forage yield may not be increased, interseeding legumes into established bermudagrass could provide an efficient way to improve pasture crude protein without the use of inorganic fertilizers. However, if alfalfa ('common’ or ‘alfagraze') is interseeded, additional P may need to be applied at legume establishment to prevent possible yield decreases.     

Both soybean and kudzu phytoestrogens modify favorably the blood lipoprotein profile in ovariectomized and castrated hamsters

The present study compared the hypolipidemic activity of kudzu phytoestrogens with that of soybean phytoestrogen in estrogen- and androgen-deficient hamsters. In the first experiment, ovariectomized hamsters (n = 37) were randomly divided into four groups (n = 9-10 each group). The first group was the control group, whereas the second group had the time-releasing estradiol-17beta subcutaneous (pellet) implants as a positive control. The third and fourth groups were orally administered soybean or kudzu phytoestrogen extracts (30 mg/kg of body weight) per day. In the second experiments, the first group of male hamsters (n = 9) received a sham operation, whereas the other three groups of male hamsters (n = 9 each) were castrated. The castrated control group received orally distilled water, whereas the second and third castrated groups were orally given 30 mg/kg soybean or kudzu phytoestrogen extracts. The results for the first experiment showed that the ovariectomized hamsters orally given soybean and kudzu phytoestrogen extracts had significantly decreased serum total cholesterol (TC) and non-high-density lipoprotein cholesterol (non-HDL-C) with HDL cholesterol (HDL-C) being unaffected. The data from the second experiment demonstrated that administration of soybean but not kudzu phytoestrogen extracts decreased significantly serum TC. However, administration of kudzu phytoestrogens caused redistribution of cholesterol among lipoproteins, leading to a significant decrease in the ratio of non-HDL-C to HDL-C. It was concluded that both soybean and kudzu phytoestrogens could modify favorably lipoprotein profiles in ovariectomized and castrated hamsters.     

Growth of perennial forage legumes in acidic soils of the Appalachian Hill‐Lands after liming

A major constraint to the renovation of forage legume‐based pastures on acidic soils of the Appalachian hill‐lands is thought to be the absence of effective rhizobia. A growth chamber experiment was done with aluminum (Al) toxic, low pH (≥ 4.2) soils from four series (Berks, Lily, Tate, and Westmoreland) that were planted with alfalfa (Medicago sativa L.), red clover (Trifolium pratense L.), white clover (Trifolium repens L.), or birdsfoot trefoil (Lotus corniculatus L.). These soils, without lime addition, were previously shown not to contain effective, naturalized populations of rhizobia for these plant species. However, a non‐toxic, pH 6.8, Watauga soil was shown to have such rhizobia but only for alfalfa. In the present study, these five soils were reexamined after liming to pH ≥ 5.5 for effective, naturalized populations of rhizobia and the efficacy of soil inoculation with commercially available rhizobia. In addition to effective, naturalized R. meliloti for alfalfa in the Watauga soil, similar populations of R. trifolii for red clover, and R. lotus for birdsfoot trefoil, were now found. Such rhizobia were also found for alfalfa in the Lily soil and for red clover in the Lily and Tate soil. Thus, liming allowed the expression of effectiveness of natural rhizobia that otherwise would not have been detected in soil pot experiments without lime. Inoculation of the toxic soils after lime addition with commercial rhizobia was effective in about half of the soil‐plant combinations that did not contain populations of effective, naturalized rhizobia. Asymbiotic shoot growth of all the plant species was significantly (P ≤ 0.05) correlated with soil pH over a range of 5.5–6.6. These results indicate that, in the absence of effective, naturalized populations of rhizobia, improvement of rhizobial inocula could increase forage production by ～34% for some species on some of the toxic soils, even after the pH of the soils is increased to ≥ 5.5.     

Legume cover crops as a nitrogen source for pecan

Crimson clover (Trifolium incarnatum L.) plus hairy vetch ( Vicia villosa Roth), red clover (Trifolium pratense L.), white clover (Trifolium repens L.), red clover plus white clover, and bermudagrass (Cynodon dactylon [L.] Pers.) were evaluated as cover crops for pecans. Crimson clover plus hairy vetch supplied the equivalent of 101 to 159 kg nitrogen (N)/ha. Red clover plus white clover supplied up to 132 kg N/ha. Either white clover or red clover alone were less effective in supplying N than when grown together. Soil Kjeldahl‐N was usually not affected or increased using the legumes compared to fertilized bermudagrass sod. Soil nitrate (NO₃) concentrations during October were occasionally higher in unfertilized legume plots than in bermudagrass plots with March‐applied N.     

Response of legumes and cereals to phosphorus in solution culture

Phosphorus deficiency is one of the Important growth limiting factors in crop production in many regions of the world. The objective of this study was to evaluate responses of alfalfa (Medicago sativa L.), red clover (Trifoilum pratense L.), common bean (Phaseolus vulgaris L.), wheat (Triticum aestivum L.) and rice (Oryza sativa L.) to concentrations of P in nutrient solution. The P treatments applied were 5, 50, 100, 200 and 400 μM P. All crop species significantly responded to an increase in external P concentrations. The optimum P concentration for maximum growth varied with crop species, but it was higher for legumes than for cereals. Rice needs minimum as red clover maximum P concentration for maximum growth in nutrient solution as compared to other crops species. Concentrations of K, Zn and Mn were significantly affected in all crop species with P addition. Suggesting positive effects of P in ameliorating Mn toxicity if this element is present in growth medium. Increasing concentrations of P in growth medium produce negative effect on K and Zn nutrition. Growth parameters and plant nutrients concentration and uptake correlation studies showed that legumes are more responsive to P fertilization as compared to cereals.     

Differential responses of forage legumes to aluminum

Response of alfalfa (Medicago sativa L.), birdsfoot trefoil (Lotus corniculatus L.), and red clover (Trifolium pratense L.) to aluminum was evaluated in a nutrient culture system under controlled conditions. In each of the species, varietal differences were also compared. In the absence of Al stress, varieties of alfalfa and Tensas red clover produced more dry weight than the other legumes. However, among the legumes tested, alfalfa was the most sensitive to Al. Aluminum reduced the uptake of many of essential nutrients. Overall, red clover cultivars experienced the least reduction in elemental uptake, whereas alfalfa cultivars experienced the greatest reduction in uptake of elements under Al stress. The efficiency ratio (ER) assisted in differentiating legumes entries into efficient and inefficient utilizers of absorbed nutrients. The ER is defined as milligrams of dry shoot weight produced per milligram of element in the shoot. The presence of Al in the growth medium reduced the ER for all elements. With a few exceptions, ER for various elements, gave positive correlations with shoot weight. The species and cultivars used in this study showed inter‐ and intraspecific differences in growth, uptake of nutrients and nutrient efficiency ratios in the presence or absence of Al stress.     

Canopy Reduction and Legume Interseeding in Irrigated Continuous Corn

Abstract

Many alternative management systems have been evaluated for corn (Zea mays L.), soybeans (Glycine max L.), and wheat (Triticum aestivum L.) production, however, most have involved rotations from one year to the next. Legume interseeding systems which employ canopy reduction in corn have not been thoroughly evaluated. One such study was initiated in 1994 at the Panhandle Research Station near Goodwell, OK, on a Richfield clay loam soil, to evaluate five legume species interseeded into established corn: yellow sweet clover (Melilotus officinalis L.), subterranean clover (Trifolium subterraneum L.), alfalfa (Medicago sativa L.), arrowleaf clover (Trifolium vesiculosum L.), and crimson clover (Trifolium incarnatum L.). In addition, the effect of removing the corn canopy above the ear (canopy reduction) at physiological maturity was evaluated. Canopy reduction increased light interception beneath the corn thus enhancing legume growth in late summer, early fall, and early spring the following year prior to planting. Forage growth from legumes incorporated prior to planting were expected to lower the amount of inorganic nitrogen (N) fertilizer needed for corn production. Crimson clover appeared to be more shade tolerant than the other species, and interseeding this species resulted in the highest corn grain yields when no N was applied. In the last two years, interseeding crimson clover at physiological maturity, followed by canopy reduction resulted in a 1.32 Mg ha^?1 increase in yield compared to conventionally grown corn with no N applied. In 1999, interseeded legumes (except subterranean clover) in conjunction with the application of 56 kg N ha^?1 and crimson clover interseeded without the addition of fertilizer N (with and without canopy reduction) resulted in grain N uptake levels equal to the 112 kg N ha^?1 treatment.     

Soy and alfalfa phytoestrogen extracts become potent low-density lipoprotein antioxidants in the presence of acerola cherry extract

Postmenopausal women have an increased risk of coronary heart disease. Oxidation of low-density lipoprotein (LDL) has been implicated in atherogenesis, and the presence of modified LDL (LDL(-)) in plasma appears to represent LDL oxidation in vivo. Because previous studies have demonstrated a strong antiatherogenic effect of estrogen due to its antioxidant activity and similar antioxidant activity was found for specific isoflavones derived from soy extract, the antioxidant activity of a phytoestrogen extract derived from soy and alfalfa was studied. Copper-mediated LDL oxidation was inhibited in the presence of soy and alfalfa extracts, and this effect was further enhanced in the presence of acerola cherry extract, which is rich in ascorbic acid. Male rabbit aortic endothelial cells pretreated with soy extract were resistant to the toxic effects of high levels of LDL and LDL(-), and a lesser, but significant protection, was also afforded by alfalfa extract. Cell-mediated oxidation of LDL, measured by LDL(-) formation, was inhibited in the presence of soy extract but not alfalfa extract. However, in the presence of acerola cherry extract, both soy and alfalfa extracts potently inhibited the formation of LDL(-). These findings show that acerola cherry extract can enhance the antioxidant activity of soy and alfalfa extracts in a variety of LDL oxidation systems. The protective effect of these extracts is attributed to the presence of flavonoids in soy and alfalfa extracts and ascorbic acid in acerola cherry extract, which may act synergistically as antioxidants. It is postulated that this synergistic interaction among phytoestrogens, flavonoids, and ascorbic acid is due to the "peroxidolitic" action of ascorbic acid, which facilitates the copper-dependent decomposition of LDL peroxides to nonradical products; this synergy is complemented by a mechanism in which phytoestrogens stabilize the LDL structure and suppress the propagation of radical chain reactions. The combination of these extracts markedly lowers the concentrations of phytoestrogens required to achieve significant antioxidant activity toward LDL.     

Different leguminous pre-crops increased yield of succeeding cereals in two consecutive years

Leguminous pre-crops are an important source of green manure in organic crop rotations for improving soil fertility and achieving high yields of cereals. We aimed to study the potential of various leguminous species, other than the traditionally cultivated red clover (Trifolium pratense L.), as green manure pre-crops for subsequent cereals. The use of different legume species enables to exploit advantages of specific legumes in organic cereal production. In order to test the legumes as pre-crops for cereals, we carried out trials located in the temperate climate zone of northeast Europe (58°44′59.41″ N, 26°24′54.02″ E). We sowed the following perennial legumes as pre-crops: red clover, alsike clover (Trifolium hybridum L.) and Washington lupine (Lupinus polyphyllus Lindl.), biennial white sweet clover (Melilotus albus Medik.) and annual Alexandria clover (Trifolium alexandrinum L.), and crimson clover (Trifolium incarnatum L.). Timothy (Phleum pratense L.) was used as a control. The leguminous pre-crops were followed by three spring cereals (barley, oat and spring wheat) and two winter cereals (rye and winter wheat). We tested the first-year after-effect (all cereals) and second-year after-effect (only barley and oat) of pre-crops on the grain yield of cereals. Perennial and biennial legume species produced the highest dry matter yield and contained the highest amount of nutrients, especially nitrogen, compared to annual species. All subsequent cereals produced significant extra yields after each leguminous pre-crop in the following two years, although the effect was smaller in the second year. The most suitable pre-crops for spring cereals were red and alsike clover followed by lupine, whereas the best pre-crops for winter cereals were sweet clover and annual clovers. Our results show the potential of various leguminous pre-crop species as valuable sources of green manure in organic crop rotation.