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Objective: Trifolium pratense has many healing properties, including fewer complications of menopause, cancer cell suppression, reducing blood glucose and lipids, as well as cardiovascular beneficial effects. The purpose of this study was to identify the phytochemical and mineral composition of T. pratense. Methods: Plant aerial parts were harvested and dried, and then hydroalcoholic and alcoholic extracts were prepared. Gas chromatography–mass spectrometry (GC–MS) analytical method was used to identify volatile compounds then liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was used to identify polyphenols and the mineral elements were identify by inductively coupled plasma atomic emission spectrometer/ICP-AES and scanning electron microscope-energy-dispersive X-ray spectroscopy (SEM-EDS) methods. Total phenolic content (TPC) was determined based on colorimetric method, and total flavonoid content (TFC) was established based on the folin-chiocalteau reagent. Furthermore, two assays (DPPH and FRAP) were used to measure the antioxidant capacity of T. pratense ethanolic extract. Results: A total of 37 polyphenols and 107 peaks were identified by LC-ESI-MS analysis, and the GC/MS method also detected 21 volatile compounds, the most important of which were methylcyclopentane, dimethylpentanal and hexadecanol. A total of 18 mineral elements, including K, Mg, Al, Si, Zn, Ni, Cu, Se, Co, Fe, Mn, and Ca in the plant, were identified ICP-AES and SEM-EDS analysis. Conclusion: T. pratense has many therapeutic compounds such as polyphenol (isoflavone and flavonoids), volatile compounds, and essential mineral elements, which can be formulated purely and used in the pharmaceutical and traditional medicine industries.
ABSTRACT
Abstract Trifolium pratense L., Fabaceae, is a rich source of isoflavones and has become the focus of several studies related to its phytoestrogenic activity. The aim of this study was to establish germination and cell cultures protocol for T. pratense and quantify isoflavones content in cell cultures, in vitro cultured and wild plants harvested in two different seasons. Murashige Skoog medium supplemented with naphthalene acetic acid and kinetin was able to produce the highest formation of friable calli. Calli cultures were analyzed qualitatively after 60 days of culture, and in vitro plants after 30, 45 and 60 days of cultivation. The chemical analysis was performed by ultra performance liquid chromatography, using the linearity curves of daidzein, genistein, formononetin and biochanin A as standards. The concentrations of isoflavones detected in wild plants were different in the two harvest periods and contrasted in content when compared to the in vitro plants. Cell cultures exhibited diverse profiles and concentration of isoflavones, none of which presented the isoflavonoid biochanin A. Pectinase was used to promote reduction of clumps and ended up altering the characteristics of secondary metabolites production in some cultures. Formononetin showed higher concentration in wild red clover samples (15.407 mg g-1), and in the in vitro grown plants the highest concentration was daidzein (17.591 mg g-1) at 60 days. The methods used for this research were effective, and the red clover plants of the analyzed variety can be cultivated in vitro aiming the commercial productivity by having contents greater than or equal to the wild plants in the periods studied, even without the use of elicitors during the cultivation.
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Abstract Background: Forage characteristics can modify in vitro methane production. There is little information about in vitro methane production of legumes and grasses at different maturity stages in tropical highland grazing systems. Objective: To evaluate the effect of species and forage maturity on in vitro methane production. Methods: Four forage species grown in tropical highlands of Colombia, two grasses: Kikuyu (Cenchrus clandestinus, previously named Pennisetum clandestinum) and ryegrass (Lolium perenne var. Samsum), and two legumes: Lotus (Lotus uliginosus var. Maku) and red clover (Trifolium pratense) were harvested in two paddocks at three maturity stages (young, intermediate, and mature). In vitro 48 h gas production was measured and methane proportion in gas was quantified by gas chromatography. Data were analysed as a randomized complete block (paddocks) design with a factorial arrangement 4×3 (4 species × 3 maturity stages) using the GLM procedure of SAS®. Results: Lotus produced less methane (p<0.01) than ryegrass, clover, and kikuyu (35.5 vs 64.7, 55.7 or 51.4 mL/g degraded organic matter, respectively). Younger forages produced less methane than intermediate and mature forages (42.8 vs 56.3 and 56.4 mL/g degraded organic matter, respectively). Cellulose concentration and organic matter degradability explained 67% (p<0.01) of methane production. Conclusion: Forage composition, presence of condensed tannins, and changes in fermentation patterns may explain the differences observed in in vitro methane production among species and maturity stages.
Resumen Antecedentes: Las características de los forrajes pueden afectar la producción de metano. Hay poca información sobre la producción de metano de leguminosas y gramíneas en diferentes estados de madurez en los sistemas pastoriles de trópico alto. Objetivo: Evaluar el efecto de la especie y la madurez de los forrajes sobre la producción de metano in vitro. Métodos: Cuatro especies forrajeras de trópico alto colombiano, dos gramíneas: Kikuyo (Cenchrus clandestinus, anteriormente llamado Pennisetum clandestinum) y ryegrass (Lolium perenne var. Samsum) y dos leguminosas: Lotus (Lotus uliginosus var. Maku) y trébol rojo (Trifolium pratense), fueron cosechadas de dos parcelas en tres estados de madurez (joven, intermedio y maduro). Se midió la producción de gas in vitro a las 48 h y la proporción de metano en el gas por cromatografía de gases. Los datos se analizaron empleando un diseño de bloques completos al azar (parcelas) con un arreglo factorial 4×3 (4 especies × 3 estados de madurez) mediante el procedimiento GLM de SAS®. Resultados: El lotus produjo menos metano (p<0,01) que el ryegrass, trébol o kikuyo (35,5 vs 64,7, 55,7 o 51,4 mL/g materia orgánica degradada, respectivamente). Los forrajes jóvenes produjeron menos metano que aquellos de edad intermedia y madura (42,8 vs 56,3 y 56,4 mL/g materia orgánica degradada, respectivamente). La concentración de celulosa y la digestibilidad de la materia orgánica explicaron el 67% (p<0,01) de la producción de metano. Conclusión: La composición de la pastura, la concentración de taninos condensados y los cambios en los patrones de fermentación pueden explicar las diferencias en la producción de metano in vitro según la especie y madurez del forraje.
Resumo Antecedentes: As caraterísticas das forragens podem modificar a produção de metano. Existe pouca informação sobre a produção de metano de leguminosas e gramíneas em diferentes fases de maturidade nos sistemas de pastoreio de trópico alto. Objetivo: Avaliar o efeito da espécie e a maturidade das forragens sobre a produção de metano in vitro. Métodos: Quatro espécies de forragens de trópico alto colombiano, duas gramíneas: Capim quicuio (Cenchrus clandestinus), antigamente chamado Pennisetum clandestinum) e azevém (Lolium perenne var. Samsum) e duas leguminosas: Trevina (Lotus uliginosus var. Maku) y trevo (Trifolium pratense), foram colhidas de duas parcelas diferentes em três fases diferentes de maturidade (jovem, intermedia e madura). Foi avaliada a produção de gás in vitro às 48 h e a proporção de metano foi determinada por cromatografia de gases. Os dados foram analisados com um delineamento em blocos ao acaso (parcelas) com um arranjo fatorial 4×3 (4 espécies × 3 fases de maturidade) utilizando o procedimento GLM de SAS®. Resultados: Trevina produz menos metano (p<0,01) que azevém, trevo o capim quicuio (35,5 vs 64,7, 55,7 ou 51,4 mL/g matéria orgânica degradada, respetivamente). Forragens jovens produzem menos metano que forragens com idade intermedia e matura (42,8 vs 56,3 y 56,4 mL/g matéria orgânica degradada, respetivamente). A concentração de celulosa e a digestibilidade da matéria orgânica explicaram o 67% (p<0,01) da produção de metano. Conclusão: A composição da pastagem, a concentração de taninos condensados e as mudanças nos padrões de fermentação podem explicar as diferencias na produção de metano in vitro entre espécies e maturidade de forragens.
ABSTRACT
Objective:To investigate the vasorelaxant effect of the isoflavones in red clover on rat mesenteric artery rings. Meth-ods:The rat mesenteric artery rings were placed between two L form metal needles. When stable precontractions induced by vasocon-strictor were got, the different concentrations of isoflavones in red clover and ( or) blocker were added to the baths. The isometric ten-sion of the rat mesenteric artery rings was recorded using a myograph system. Results:The isoflavones in red clover could concentra-tion-dependently relax the rat mesenteric artery rings pre-contracted by K+krebs solution (60 mmol·L-1K+), and compared with the control group (Dmso), the difference was statistically significant (P<0. 01). The isoflavones in red clover-induced relaxation was re-duced by the removal of endothelium, and compared with the complete endothelial group, the difference was statistically significant ( P<0. 01). Pretreatment with Nω-nitro-L-arginine-methylesterhydrochloride (L-NAME,10 -4 mol·L-1) could suppress the relaxing re-sponse to the isoflavones in red clover, and compared with the control group, the difference was statistically significant (P<0. 01). However, indomethacin(10 -5 mol·L-1) did not significantly affect the relaxation induced by the isoflavones in red clover (P >0. 05). Conclusion:The vasorelaxant effect of the isoflavones in red clover is through promoting nitric oxide release from vascular en-dothelium.
ABSTRACT
Objective:To investigate the vasorelaxant effect of the isoflavones in red clover on rat mesenteric artery rings. Meth-ods:The rat mesenteric artery rings were placed between two L form metal needles. When stable precontractions induced by vasocon-strictor were got, the different concentrations of isoflavones in red clover and ( or) blocker were added to the baths. The isometric ten-sion of the rat mesenteric artery rings was recorded using a myograph system. Results:The isoflavones in red clover could concentra-tion-dependently relax the rat mesenteric artery rings pre-contracted by K+krebs solution (60 mmol·L-1K+), and compared with the control group (Dmso), the difference was statistically significant (P<0. 01). The isoflavones in red clover-induced relaxation was re-duced by the removal of endothelium, and compared with the complete endothelial group, the difference was statistically significant ( P<0. 01). Pretreatment with Nω-nitro-L-arginine-methylesterhydrochloride (L-NAME,10 -4 mol·L-1) could suppress the relaxing re-sponse to the isoflavones in red clover, and compared with the control group, the difference was statistically significant (P<0. 01). However, indomethacin(10 -5 mol·L-1) did not significantly affect the relaxation induced by the isoflavones in red clover (P >0. 05). Conclusion:The vasorelaxant effect of the isoflavones in red clover is through promoting nitric oxide release from vascular en-dothelium.
ABSTRACT
Red clover is one of the most utilized forage in agriculture and contains many of the isoflavones known for their human health benefits. The objectives of this study were: i) to quantify, using HPLC analysis, isoflavones in 77 accessions from the USDA core collection and a Brazilian line; ii) to verify possible relationships depending on their origin, improvement status or maturity type and; iii) to verify the seasonal variation. The isoflavone mean contents were 29.27 µg g-1 of dry material for daidzein, 163.69 µg g-1 for genistein, 11353.29 µg g-1 for formononetin and 6568.8 µg g-1 for biochanin A. Clustering was mainly influenced by the total amount of isoflavones and partially due to maturity type, improvement status and geographic origin. The seasonal evaluation demonstrated an increase of concentration during winter, and decrease during spring. These results highlighted accessions that can be used to develop new varieties with low or high isoflavones concentration.
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Isoflavones are polyphenolic phytoestrogens, predominantly found in leguminous plants. Trifolium pratense L., Fabaceae (red clover), is rich in isoflavones that possess estrogenic activity due to their similar molecular structure and effectiveness in preventing health conditions such as menopause, osteoporosis, cardiovascular disease, hypertension and hormone-dependent cancers. In this study, presence and amount of various phytoestrogens in the tetraploid plant and in the calluses derived from the plants were investigated. Calluses were generated from explants obtained from natural tetraploid T. pratense seedlings. The best callus formation was obtained from hypocotyl explants cultured in Phillips Collins and Gamborg B5 media containing different plant growth regulators. Flowers of plants and calluses were analysed for formononetin, biochanin A, genistein and daidzein contents using HPLC. In HPLC analysis, high levels of formononetin (0.249 µg/mg) were determined in natural tetraploid T. pratense flowers in addition to genistein and biochanin A. In calluses, highest isoflavone content (1.15 µg/mg formononetin) was observed in modified Gamborg B5 medium. Biochanin A content of calluses and the plant were found to be nearly the same. But formononetin and genistein contents of the calluses in this medium were found to be respectively 4.62 and 21.39 folds higher than the tetraploid plant.
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This study was designed to investigate the potential estrogenic effects of perinatal dietary phytoestrogens on the rat uterus. Pregnant rats were divided to three groups provided the following diets: (1) rat chow, (2) rat chow with 7.5% Trifolium (T.) pratense, or (3) rat chow supplemented with 17beta-estradiol (0.5 mg/kg). The dams in each group were kept on the same diet during pregnancy and lactation. Female offspring were euthanized on day 21 at which time body and organ weights were recorded and tissue samples were taken for histology. Immunohistochemistry was performed to detect estrogen receptor alpha (ERalpha) and progesterone receptor (PR) levels. Our results revealed estrogen-like biological effects of perinatal T. pratense exposure. Relative uterus and ovary weights in the experimental groups were increased compared to control. The number of uterine glands and luminal epithelium heights were also increased. However, there were no statistically significant changes detected in the immunostaining intensity of ERalpha and PR between the groups.
Subject(s)
Animals , Female , Pregnancy , Rats , Animals, Suckling , Body Weight/drug effects , Estrogen Receptor alpha/metabolism , Immunohistochemistry , Isoflavones/pharmacology , Lactation , Maternal Exposure , Organ Size/drug effects , Phytoestrogens/pharmacology , Plant Components, Aerial/chemistry , Rats, Wistar , Receptors, Progesterone/metabolism , Trifolium/chemistry , Uterus/drug effectsABSTRACT
The study investigated the effects of red clover extract (RCE) on mouse T macrophages and lymphocytes in vitro. The cell toxic effect of RCE was estimated by MTT assay. Multiple-fluorescence staining plus flow cytometry were used to detect the effect of RCE on CD69/CD25/CD71 expression of mouse T lymphocytes stimulated by Con A; CFDA-SE staining plus flow cytometry were used to analyze the effect of RCE on proliferation of T lymphocytes activated by Con A; The effect of RCE on nitric oxide(NO) secretion of mouse macrophages stimulated by lipopelysaccharide (LPS) for 24 h was assayed by Griess reagent system. We found that RCE had potent anti-inflammatory effects on mice. RCE had littl ecell toxic effect on mouse lymphocytes and macrophages. RCE strongly inhibited the excessive production of inflammatory mediators ( NO, CD69, CD25, CD71 ), in a dose-dependent manner, like cyclosporine A injection. RCE could inhibit proliferation of CD3<'+> T lymphocytes. These data suggested that RCE migh texhibit anti-inflammatory effect by inhibiting the activation and proliferation of mouse lymphocytes and the NO secretion of mouse macrophages.
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The regeneration of natural tetraploid T. pratense, originated from Erzurum-Turkey, is reported in this study. This plant has low seed setting and hard seed problems due to polyploidy. Hypocotyl, cotyledon, apical meristems, epicotyl and young primary leaves were inoculated on MS and PC-L2 media containing different concentrations of BAP and NAA as growth regulators. The best shoot formation has been observed on explants initiated from apical meristem placed on PC-L2 medium that includes 2 mg dm-3 BAP and 1 mg dm-3 NAA. 94.4 percent of the shoots originated from calli were rooted on PC-L2 medium with 1 mg dm-3 NAA. In vitro organogénesis has been accomplished in the natural tetraploid T. pratense regenerated plants successively transferred to the field.