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A simplification of a haemolytic micromethod is presented. In the orginal method, alfalfa (Medicago media Pers.) leaf sap is spotted on the plate covered with a blood-gelatine suspension. In the presented method, the mashed alfalfa pulp is used instead of sap. Due to saponin diffusion and the reaction with erythrocytes, a haemolytic ring appears, which has a width proportional to the concentration of toxic saponins. It is shown that the width of haemolytic ring does not depend on the sample weight ranging from 20 to 100 mg. This allows for the omission of laborious sap pressing and sample quantification. Individual alfalfa plants with different saponin contents were tested using leaf sap and leaf pulp for analyses. Good agreement was found with sap and leaf pulp methods. The correlation obtained by both methods was high, r = 0.87. The modified method requires only a small amount of plant material and makes the analyses of large numbers of individual plants per day possible. The method is especially recommended for breeding purposes.
Different concentrations of medicagenic acid and five glycosides of this acid isolated from alfalfa (Medicago sativa) were added to agar medium (com meal agar, CMA) inoculated with cultures of Gaeumannomyces graminis var. tritici (Ggt). After 7 days of incubation at 25°C colony radius was measured and % of inhibition calculated in relation to the control medium (CMA enriched with the solvent of the tested compounds). Within the tested concentrations, only 3-O-β -D -glucopiranoside medicagenate (monoglucoside) significantly reduced the growth of Ggt on CMA medium. This compound at 0.05 mM concentration completely inhibited the development of the fungus and the effect was shown to be fungi-toxic.
In the present work it was shown that total saponins originated from M. hybrida and M. sativa substantially limited mycelium growth of F. oxysporum f. sp. tulipae and symptoms of fusariosis on tulip bulbs. Out of 15 individual tested saponins originated from M. arabica, M. hybrida and M. sativa, four compounds: 3-O-[β-D-glucopyranosyl (1→2)α-L-arabinopyranosyl] hederagenin, hederagenin 3-O-β-D-glucopyranoside, medicagenic acid, medicagenic acid 3-O-β-D-glucopyranoside had the strongest inhibitory effect on mycelium growth of Fusarium oxysporum f. sp. tulipae on PDA medium. The total saponins from M. arabica, M. hybrida and M. sativa inhibited the number of colony forming units of Fusarium oxysporum f. sp. tulipae in artifi cially infested substrate. The use of saponins originated from Medicago as a fungicide is suggested.
Preliminary investigations were carried out in order to ascertain the occurrence of isoflavonoid compounds characterized by estrogenic activity, in five domestic species of clover: Trifolium pratense L., T. repens L., T. incurnatum L., T. resupinatum L. and T. hybridum L. Large amounts of biochanin A (0.8% d.m.) and formononetin (0.7% d.m.) and small amounts of genistein were identified in the green parts of red clover. In the remaining species, small amounts of formononetin and genistein were found. In all the species of clover, free isoflavonoid aglycones and glycosides were found. Laboratory tests carried out on mice showed that only T. pratense contained high estrogen activity. Red clover also had an unidentified factor which inhibited the increase of the body weight of young mice.
The concent of formononetin, the predominant estrogen, was analysed in four cultivars of red clover. Its highest content was found in cv. Jubilatka (tetraploid) - 0,57 % DM, next, in cv. Hruszowska (diploid) - 0,44%, Radyka (tetraploid) - 0,39 % and Skrzeszowicka (diploid) - 0,3 %. High variation from plant to plant was found in all of the analysed cultivars, which makes it possible to select for a low formononetin clover population. The content of formononetin in individual plants in two consecutive years was positevely correlated. The content of biochemin A in two cultivars was also analysed employing a semi-quantitative TCL test. It was found that the concent of these two estrogens in individual plants was positively correlated. However, some individual plants containing high ammount of only one estrogen were also found. Because of that in the selection for low-estrogen red clover, after selection of low formononetin plants, an additional test for biochanin A is recomended.
The total saponins of alfalfa, Medicago sativa L., included in the diet of Colorado potato beetle larvae reduced their feeding, growth rate and survival. The biological activity of those compounds coming both from the roots and from the aerial parts is ,y correlated with the dose. Larvae reared on leaves treated with a 0.5% dose virtually did not feed at all and died after 4-6 days. Lower saponin doses (0.0 I and %) reduced the insects - feeding to a lesser degree. However, they inhibited their ,caused an extension of the larval stage and mortality at a level of76.7- 100%. No major differences have been found in saponin activity depending on its localization in the plant.
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