Phytochemicals and antioxidant activity of alcoholic/hydroalcoholic extract of Trifolium pratense / 中草药·英文版

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.

Canopy structure and morphogenesis of Italian ryegrass intercropped with red clover under cutting intervals determined by thermal sum / Estrutura do dossel e morfogênese do azevém anual consorciado com trevo vermelho em intervalos de corte determinados por soma térmica

ABSTRACT: The canopy structure and morphogenesis of ryegrass (Lolium multiflorum Lam.) was investigated after it was over seeded with Tifton 85 (Cynodon spp.) and intercropped with red clover (Trifolium pratense L.). Time intervals between cuts were determined by different thermal sums (125, 250, 375, and 500 degree-days [DD]). Cuts were conducted at a height equivalent to 50% of the average canopy height. Morphogenic and structural data were collected from July to October of 2016 and 2017, from a completely randomized block experimental design. Canopy height, extended tiller height, pseudostem length, and forage mass were fitted to linear equations according to the thermal sums. Interaction of ryegrass with red clover was the highest (12.1%) for longer cut intervals (375 and 500 DD). The leaf appearance rate (0.0080 leaf/DD), phyllochron value (128.8 DD), and senescence rate (0.10 cm/DD) of ryegrass were comparable between the different intervals. The slowest elongation rate (0.06 cm/DD) with the shortest interval duration between cuts (171.6 DD) was observed in the 125 DD interval. According to the findings from this 2-year experiment, the use of a 250 DD cut interval was recommended between grazing events, taking into account the efficiency of forage use, ryegrass leaf, stem ratio, and interactions with red clover in the canopy under rotational stocking.

RESUMO: A estrutura do dossel e a morfogênese do azevém (Lolium multiflorum Lam.) foram investigadas após a sobressemeadura em Tifton 85 (Cynodon spp.) e consorciado com trevo vermelho (Trifolium pratense L.). Os intervalos de tempo entre os cortes foram determinados por diferentes somas térmicas (125, 250, 375 e 500 graus-dia [GD]). Os cortes foram realizados a uma altura equivalente a 50% da altura média do dossel. Os dados morfogênicos e estruturais foram coletados no período de julho a outubro de 2016 e 2017, em um delineamento experimental de blocos ao acaso. A altura do dossel, a altura estendida do perfilho, o comprimento do pseudocolmo e a massa de forragem se ajustaram a equações lineares de acordo com a soma térmica. A consorciação de azevém e trevo vermelho foi maior (12,1%) para intervalos de corte mais longos (375 e 500 GD). A taxa de aparecimento foliar (0,0080 folha/GD), filocrono (128,8 GD) e a taxa de senescência (0,10 cm /GD) do azevém não foram diferentes entre os intervalos de corte. A menor taxa de elongação (0,06 cm/GD) e duração de elongação (171,6 GD) foi observada no menor intervalo entre cortes, 125 GD. De acordo com os achados deste experimento de dois anos, recomenda-se o uso de um intervalo de corte de 250 GD entre os eventos de pastejo, levando em consideração a eficiência do uso de forragem, proporção de folhas e colmo de azevém e trevo vermelho no dossel, sob lotação rotativa.

Trifolium pratense induces apoptosis through caspase pathway in FaDu human hypopharynx squamous carcinoma cells

Trifolium pratense leaves (red clover) has been used in Oriental and European folk medicine for the treatment of whooping cough, asthma, and eczema, and is now being used to treat and alleviate the symptoms, such as hot flushes, cardiovascular health effects that occur in postmenopausal women. However, relatively little scientific data is available on the physiological activity of this plant. Therefore, in this study, we investigated the anti-cancer activity of T. pratense leaves using methanol extract of T. pratense leaves (MeTP) on human FaDu hypopharyngeal squamous carcinoma cells. MeTP inhibited the viability of FaDu cells by inducing apoptosis through the cleavage of procaspase-3,

Analysis and Comparison of Volatile Components in Trifolium pratense and Trifolium repens Seed by GC-MS / 中国实验方剂学杂志

Objective: To analyze and compare the difference of volatile components between Trifolium pratense and T. repens seeds.Method: The volatile components were extracted by the headspace solid phase microextraction (HS-SPME) method and analyzed by gas chromatography-mass spectrometry (GC-MS),and the relative contents of the components were calculated by the area normalization method.Result: Fourty six compounds were identified in the seeds of T. pratense,with a total peak area of 3.48×109 for the volatile components. The compounds with high content in this species were 1-octen-3-ol (30.24%),1-hexanol (11.94%),heptane,2,2,4,6,6-pentamethyl (11.00%),nonanal (7.74%),octanal (6.79%) and hexanoic acid (6.04%). 44 compounds were identified from the seeds of T. repens,with a total peak area of 6.37×109 for the volatile components. The compounds with high content in this species were 1-octen-3-ol (42.98%),heptadecane (14.47%),3-octanol (9.42%),n-caproic acid vinyl ester (8.82%) and pyrimidine-4,6-diol,5-methyl (6.59%). 20 components were commonly shared in the seeds of T. pratense and T. repens,but the content of each component was quite different. 1-octen-3-ol had the highest content in the common components. T. pratense seeds had 26 unique components,accounting for 54.85%of volatile components. T. repens seeds had 24 unique components,accounting for 46.79%of volatile components.Conclusion: The seeds of both T. pratense and T. repens have rich volatile components,but there are great difference in the variety,peak area and relative content of the volatile components between these two varieties

Differential formononetin content in cultivars and experimental lines of red clover (Trifolium pratense L. ) plants affect the feeding behaviour of Hylastinus obscurus (Coleoptera: Curculionidae) / Contenido diferencial de formononetina en cultivares y líneas experimentales de plantas de trébol rosado (Trifolium pratense L. ) afecta la conducta alimenticia de Hylastinus obscurus (Coleoptera: Curculionidae)

Red clover (Trifolium pratense L.) is a perennial plant widely used as a forage resource for several animals. This plant is the exclusive host of Hylastinus obscurus (Marsham) which causes irreparable damages to the root system affecting their persistence. It has been reported that the presence of the isoflavonoid formononetin in roots of red clover could act as an antifeedant on H. obscurus. There are not studies related to the formononetin content in red clover roots to the antifeedant effect elicited by experimental lines and cultivar of red clover. Six red clover genotypes were investigated in both formononetin content and their respective antifeedant action. The results showed to Sabtoron High and Superqueli-INIA with both the highest formononetin content in red clover roots and antifeedant effect, allowing to suggest that this secondary metabolites could be used as a chemical factor for red clover plants. Moreover, a rapid methodology for searching red clover genotypes with high formononetin content is reported.

El trébol rosado (Trifolium pratense L.) es una planta perenne ampliamente utilizada como fuente de forraje de variados animales. Esta planta es el exclusivo hospedero de Hylastinus obscurus (Marsham) el cual causa irreparables daños al sistema radical afectando seriamente su persistencia. Se ha reportado que la presencia del isoflavonoide formononetina en raíces del trébol rosado podría actuar como antialimentario sobre H. obscurus. Actualmente no existen estudios que relacionen el contenido de formononetina en raíces de trébol rosado con el efecto antialimentario elicitado por líneas experimentales y cultivares de trébol rosado. Seis genotipos de esta leguminosa fueron evaluados en cuanto a su contenido de formononetina y actividad antialimentaria. Los resultados mostraron que los cultivares Sabtoron High y Superqueli-INIA presentaron altos niveles de formononetina en sus raíces y efecto antialimentario sobre H. obscurus, lo que permite sugerir que este metabolito secundario podría ser usado como factor químico para incrementar la persistencia de plantas de trébol rosado. Además, se informa una metodología rápida para la búsqueda de genotipos con altos contenidos de formononetina.

Efficient discovery and capturing of nNOS-PSD-95 uncouplers from Trifolium pratense / 中国中药杂志

Magnetic molecularly imprinted polymers(MMIPs) were prepared with ZL006 as template, acrylamide(AA) as the functional monomer, and acetonitrile as pore-forming agent; then Fourier transform infrared spectroscopy(FT-IR) and scanning electron microscopy(SEM) were used to characterize their forms and structures. Simultaneously, the MMIPs prepared previously were used as sorbents for dispersive magnetic solid phase extraction(DSPE) to capture and identify potential nNOS-PSD-95 uncouplers from extracts of Trifolium pratense and the the activities of the screened compounds were evaluated by the neuroprotective effect and co-immunoprecipitation test. The experiment revealed that the successfully synthesized MMIPs showed good dispersiveness, suitable particle size and good adsorption properties. Formononetin, prunetin and biochanin A were separated and enriched from Trifolium pratense by using the MMIPs as artificial antibodies and finally biochanin A was found to have higher cytoprotective action and uncoupling action according to the neuroprotective effect and co-immunoprecipitation test.

Forage yield and nutritive value of Elephant grass, Italian ryegrass and spontaneous growing species mixed with forage peanut or red clover / Massa de forragem e valor nutritivo de capim elefante, azevém e espécies de crescimento espontâneo consorciadas com amendoim forrageiro ou trevo vermelho

The objective of this research was to evaluate of three grazing systems (GS) with elephant grass (EG), Italian ryegrass (IR) + spontaneous growing species (SGS); EG + IR + SGS + forage peanut (FP); and EG + IR + SGS + red clover (RC), during the winter and summer periods in rotational grazing with dairy cattle. Experimental design was completely randomized with three treatments, two replicates with repeated measures. Lactating Holstein cows receiving 1% BW-daily feed supplement with concentrate were used in the evaluation. Eight grazing cycles were performed during the experimental period. The values of pre forage mass and stocking rate were 2.52, 2.60 and 2.99 t ha-1 and 2.64, 2.77 and 3.14 animal unit ha-1, respectively for GS. Samples of forage were collected by hand-plucking technique to analyze the crude protein (CP), neutral detergent fiber (NDF), in situ dry matter digestibility (ISDMD), in situ organic matter digestibility (ISOMD) of forage present between rows of elephant grass, in the rows of elephant grass and the legumes. Higher value of CP, ISOMD and lower of NDF were observed for the grazing systems mixed with legumes forage.

O objetivo desta pesquisa foi avaliar três sistemas forrageiros (SF) com capim elefante (CE) + azevém (AZ) + espécies de crescimento espontâneo (ECE); CE + AZ + ECE + amendoim forrageiro (AM); e CE + AZ + ECE + trevo vermelho (TV), durante os períodos hibernal e estival, em pastejo rotativo com bovinos leiteiros. O delineamento experimental foi o inteiramente casualizado, com três tratamentos, duas repetições, com medidas repetidas no tempo. Para avaliação foram usadas vacas da raça Holandesa em lactação, que receberam suplementação alimentar à razão de 1% do peso corporal dia-1. Durante o período experimental foram efetuados oito ciclos de pastejo. Os valores de massa de forragem de pré-pastejo e da taxa de lotação foram de 2,52; 2,60 e 2,99 t ha-1 e 2,64; 2,77 e 3,14 UA ha-1, respectivamente para os SF. Foram coletadas amostras de forragem, simulando o pastejo, para determinação dos teores de proteína bruta (PB), fibra em detergente neutro (FDN), digestibilidade in situ da matéria seca (DISMS) e digestibilidade in situ da matéria orgânica (DISMO) da forragem presente entre as linhas de capim elefante, da linha de capim elefante e das leguminosas. Foram observados maiores valores de PB, DISMO e menores de FDN para os sistemas consorciados com leguminosas.

Effect of Trifolium pratense extract on methionine-choline-deficient diet-induced steatohepatitis in C57BL/6 mice / 中国天然药物

AIM@#The potential of Trifolium pratense (red clover) extract in the prevention of lipid disorder has attracted increasing attention in recent years. In this study, the aim was to determine whether and how red clover extract affected the development of murine diet-induced non-alcoholic steatohepatitis.@*METHODS@#Non-alcoholic steatohepatitis was induced in C57BL/6 mice by feeding mice with a methionine-choline-deficient (MCD) diet. Hematoxylin and eosin staining was used for histological analyses. Real-time PCR was used to analyze the mRNA expression levels.@*RESULTS@#Hepatic steatosis and necroinflammation was observed in MCD diet-fed mice, and this diet-induced steatosis was significantly attenuated, whereas liver inflammation was not significantly attenuated, by red clover extract treatment. Consistent with the results of H&E staining, the MCD diet-induced increase of liver triglycerides and cholesterol levels were significantly reduced by red clover extract treatment. However, with the improvement in hepatic steatosis, mRNA levels of acetyl CoA oxidase, carnitine palmitoyl transferase-1, and liver fatty acid-binding protein, three genes regulated by peroxisome proliferator-activated receptor (PPAR) α, were unaffected.@*CONCLUSION@#Red clover extract alleviated MCD diet-induced hepatic steatosis, but did not ameliorate liver inflammation in C57BL/6 mice, and the improvement in hepatic steatosis was not through activating PPARα.

Produtividade de sistemas forrageiros consorciados com leguminosas / Productivity of grazing systems mixed with forage legumes

O objetivo desta pesquisa foi avaliar três sistemas forrageiros (SF) com capim elefante (CE) + azevém (AZ) + espécies de crescimento espontâneo (ECE); CE + AZ + ECE + amendoim forrageiro (AM); e CE + AZ + ECE + trevo vermelho (TV), usando-se a mesma área, sob pastejo rotacionado, no decorrer do ano agrícola. O CE foi estabelecido em linhas afastadas a cada 4m. No período hibernal, fez-se o estabelecimento do AZ entre as linhas do CE; o TV foi semeado e o AM foi preservado, considerando-se os respectivos SF. O delineamento experimental foi o inteiramente ao acaso, com três tratamentos (SF), duas repetições (piquetes) e avaliações independentes (ciclos de pastejos). Para avaliação, foram utilizadas vacas da raça Holandesa em lactação, que receberam suplementação alimentar com concentrado à razão de 1% do peso corporal/dia. Foram avaliados a massa de forragem, os componentes botânicos do pasto e estruturais do CE e a taxa de lotação. Durante o período experimental, foram efetuados oito ciclos de pastejo. Sistemas forrageiros que envolvem gramíneas e leguminosas de diferentes ciclos proporcionam a utilização da área durante todo o ano agrícola em pastejo rotativo com bovinos leiteiros. Considerando-se a predominância das avaliações em cada pastejo, os sistemas forrageiros consorciados apresentam melhor resultado tanto paras as variáveis de massa de forragem quanto para a taxa de lotação.

The objective of this research was to evaluate three grazing systems (GS) with elephant grass (EG) + ryegrass (RG) + spontaneous growing species (SGS); EG + RG + SGS + forage peanut (FP); and EG + RG + SGS + red clover (RC), in order to use the area in rotational grazing during the agricultural year. EG was planted in rows with a distance of 4m between each row. In the cool-season, RG was sowed between EG rows and FP was preserved on GS. The experimental design was completely randomized with three treatments (GS), two replicates (paddocks) and independent evaluations (grazing cycles). For the evaluation lactating Holstein cows receiving 1% of BW/day feed supplement concentrate were used. The herbage mass, botanical composition of pasture, structural component of EG and stocking rate were evaluated. Eight grazing cycles were performed during the experimental period. Grazing systems involving grass and legume forage in different cycles provided the use of the area during the agricultural year in rotational grazing with dairy cattle. Considering the predominance of the evaluations in each grazing, the mixed grazing systems have better results for both forage mass and stocking rate variables.

Produtividade e proteína bruta de pastagens de capim elefante manejadas sob os sistemas agroecológico e convencional / Productivity and crude protein of elephant grass pastures managed under agroecological and conventional systems

O objetivo desta pesquisa foi avaliar a massa, o teor de proteína bruta da forragem e a taxa de lotação em pastagens de capim elefante em dois sistemas de produção. No sistema convencional, o capim elefante foi plantado em linhas afastadas a cada 1,2m. No sistema agroecológico, o capim foi plantado em linhas afastadas a cada 3m; no espaço entre as linhas, no período hibernal, foram semeados o azevém anual e o trevo vermelho e, no período estival, permitiu-se o desenvolvimento de espécies de crescimento espontâneo. Foram utilizados 150kg de N ha-1 com fertilizantes químico e orgânico para os respectivos sistemas forrageiros. Foram usadas vacas em lactação que receberam complementação à razão de 0,9% do peso vivo. Para determinação do teor de proteína bruta, foram retiradas amostras simulando o pastejo. Valores similares foram observados quanto à composição estrutural do capim elefante em ambos os sistemas. Durante o período experimental, foram conduzidos oito e sete pastejos para os respectivos sistemas. Os valores médios de massa de forragem de pré-pastejo e da taxa de lotação foram de 3,7 e 4,8t ha-1 e de 2,10 e 2,51UA ha-1 dia-1. A pastagem sob sistema agroecológico apresenta melhor distribuição de forragem ao longo do ano. Teores elevados de proteína bruta foram observados no período hibernal, em ambos os sistemas forrageiros.

The objective of this research was to evaluate the forage mass, crude protein and stocking rate on elephantgrass pastures in two production systems. In the conventional system elephantgrass was planted in rows 1,2m apart from each other. In the agroecological system elephant grass was planted in rows 3m apart from each other. In the space between alleys, in cool season, annual ryegrass and red clover were introduced; allowing the development of spontaneous growing species in the warm season. It was applied 150kg of N ha-1 with chemical and organic fertilizers in the respective pastures. Holstein cows receiving 0.9% of body weight complementary concentrate feed were used. Hand-plucked samples were collected to analyze crude protein. Similar value was found in the structural components of elephantgrass pastures in both pasture systems. Eight and seven grazing cycles were performed during the experimental period for respective systems. The average dry matter value for pre-grazing and stocking rate were 3.7 e 4.8t ha-1 and 2.10 e 2.51AU ha-1 day-1 for each respective systems. Agroecological system presents best forage distribution throughout the year. Higher values of crude protein of elephantgrass were observed in cool season in both systems.

Produtividade de sistemas forrageiros consorciados com amendoim forrageiro ou trevo vermelho / Productivity of pastures-based systems mixed to forage peanut or red clover

O objetivo desta pesquisa foi avaliar três sistemas forrageiros (SF) com capim elefante (CE) Pennisetum purpureum Schum., cv. 'MerckeronPinda' + espécies de crescimento espontâneo (ECE) + azevém anual (AZ) Lolium multiflorum Lam., cv. 'Comum', como SF1; CE + ECE + AZ + amendoim forrageiro (AF) Arachis pintoi Krap. e Greg., cv. 'Amarillo', como SF2; e CE + ECE + AZ + trevo vermelho (TV) Trifolium pratense L., cv. 'Estanzoela 116', como SF3. O CE foi estabelecido em linhas afastadas a cada 4m. O azevém anual foi estabelecido entre as linhas do CE durante o período hibernal; o TV foi semeado e o AF foi preservado nos respectivos tratamentos. Para avaliação, foram usadas vacas da raça Holandesa que receberam 5,5kg dia-1 como complemento alimentar. Foram avaliadas a taxa de acúmulo diário de matéria seca (TA), a massa de forragem desaparecida (MFD), a matéria seca desaparecida com base em 100kg de peso vivo (MSD) e a produção de forragem (PF), as composições botânica e estrutural do CE. O delineamento experimental foi o inteiramente casualizado, com três tratamentos (SF) e duas repetições (piquetes) em parcelas subdivididas no tempo (pastejo). Durante o período experimental (341 dias), foram efetuados nove ciclos de pastejo. Os valores médios de TA, MFD, MSD e PF foram de 53,16kg ha-1; 36,13%; 2,77kg de matéria seca por 100kg de peso vivo e 17,80t ha-1. Para a variável ECE, houve aumento significativo no SF1. Considerando a carga animal, o SF3 apresentou melhor desempenho.

The objective of this research was to evaluate of tree pasture-based systems (PS) with elephantgrass (EG) Pennisetum purpureum Schum., cv. 'Merckeron Pinda' + spontaneous growing species (SGS), annual ryegrass (RG) Lolium multiflorum Lam., cv. 'Comum', for PS1; EG + SGS + forage peanut (FP) Arachis pintoi Krap. e Greg., cv. 'Amarillo', for PS2; and EG + SGS + RG + red clover (RC) Trifolium pratense L., cv. 'Estanzoela 116', for PS3. EG was planted in lines with a distance of 4m each one of them. Ryegrass was sowed between rows of EG during the cool-season; red clover was sowed and the forage peanut was preserved on respectively treatments. Holstein cows receiving 5.5kg daily-1 complementary concentrate feed were used. The dry matter daily accumulation rate (DMA),disappearance of forage mass (DFM),dry matter disappeared based on 100kg of live weight (DMD),total dry matter production (TDM), botanical composition and structural component of EG were evaluated. The experimental design used was completely randomized with tree treatments (SF) and two replicates (paddocks) in incomplete split-plot time (grazing cycles). Nine grazing cycle were performed during the experimental period (341 days). The average values of DMA, DFM, DMD and TDM were 53.16kg ha-1, 36.13%, 2.77kg of dry matter per100kg of liveweight and 17.80t ha-1, respectively. SGS parameter increased significantly over in the PS1. Considering the stocking rate the PS3 showed a better performance.

In vitro isoflavonoid production and analysis in natural tetraploid Trifolium pratense (red clover) calluses

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.

In vivo and in vitro anti-inflammatory activity of red clover Trifolium pratense dry extract

Red clover Trifolium pratense L., Fabaceae, contains four isoflavones, mainly formononetin and biochanin A, and in smaller concentrations, daidzein and genistein. These compounds have gained a lot interest due to its human health benefits, such as estrogenic and progestogenic activities, antioxidant, anti-cancer and others. The objective of this study was to determine in vivo and in vitro anti-inflammatory activity of red clover dry extract. The in vitro anti-inflammatory activity was assayed by the technique using the Boyden chamber method, evaluating the leukocyte migration inhibition (chemotaxis). The in vivo anti-inflammatory activity was tested by a carrageenan-induced rat paw edema test. The results of anti-inflammatory in vitro test showed that there was a significant inhibition of leukocyte migration at the concentrations of 100, 50, 25, 10 and 5 µg/mL of red clover dry extract, these doses resulted in 94.73, 95.39, 94.73, 84.68 and 78.75 percent of inhibition for each dose, respectively. The anti-inflammatory in vivo test resulted in a significant activity in both tested doses (100 and 50 mg/kg of red clover dry extract) and at each tested time. The average percentage of edema inhibition was 63.37 percent. The findings of this study suggested that red clover extract might be suitable for the treatment of inflammatory diseases.

Effect of Trifolium pratense isoflavones on osteoporosis rats induced by estrogen deficiency / 中国药学杂志

OBJECTIVE: To examine the effects of Trifolium pratense isoflavones (TPIF), the main active component of Trifolium pratense L., on ovariectomy-induced osteoporosis in rats. METHODS: A total of 60 six-month-old female rats were randomly assigned to a sham-operated group and five ovariectomized(OVX) groups, ie, OVX with vehicle(10 mL · kg-1 · d-1, ig), OVX with graded doses of TPIF(30, 60, and 90 mg · kg-1 · d-1, ig), and OVX with nilestriol(2.5 mg · kg-1 · week-1, ig). The animals were sacrificed after 12-week treatment. RESULTS: Compared to the OVX group, TPIF significantly inhibited the increase of body weight. In addition, TPIF administration significantly decreased serum levels of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), and increased serum level of estrogen. CONCLUSION: TPIF may be a potential alternative medicine for the prevention and treatment of postmenopausal osteoporosis. Its mechanism may be related to increasing estrogen level, inhibiting bone resorption, and promoting bone formation.

Evaluation of the estrogenic effects of dietary perinatal Trifolium pratense

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.

Efeitos do Trifolium pratense nos sintomas climatéricos e sexuais na pós-menopausa / Effects of Trifolium pratense on the climacteric and sexual symptoms in postmenopause

OBJETIVO: Avaliar os efeitos do tratamento com Trifolium pratense nos sintomas climatéricos e na satisfação sexual de mulheres na pós-menopausa. MÉTODOS: Este estudo foi prospectivo, randomizado, duplo-cego e controlado com placebo. Foram selecionadas 120 mulheres na faixa etária de 45 anos a 65 anos com sintomas climatéricos, amenorreia superior a um ano e sem tratamento nos últimos seis meses. Após a seleção, foram divididas em dois grupos: GT -receberam Trifolium pratense na dose de 40 mg, 1 capsula/dia; GP -receberam placebo (controle), contendo lactose, 1 cápsula/dia. A duração do tratamento foi de 12 meses. As pacientes foram avaliadas clinica e laboratorialmente antes do tratamento e com quatro, oito e 12 meses de tratamento. Foi empregado também o Índice Menopausal de Kupperman (IMK) e o Inventário de Satisfação Sexual Golombok Rust. No final do estudo, cada grupo tinha 50 pacientes. RESULTADOS: Houve melhora significante dos sintomas menopausais após quatro meses de tratamento pelo IMK, principalmente em relação aos fogachos, comparando os dados antes do tratamento nos dois grupos, porém, não houve diferença significante entre os grupos. Não houve melhora na sexualidade antes e após o tratamento. CONCLUSÃO: O tratamento por 12 meses com Trifolium pratense, na dose de 40mg/dia, não promoveu melhora significante dos sintomas menopausais e na satisfação sexual.

OBJECTIVE: To evaluate effects of treatment with Trifolium pratense on climacteric symptoms and sexual satisfaction in postmenopausal women. METHODS: This study was prospective, randomized, double-blind and placebo-controlled. We selected 120 women, aged between 45 and 65 years with climacteric symptoms, with absence of menstruation (amenorrhea) for more then one year and without any treatment in the last six months. After selection, women were divided into two groups: GT received 40 mg of Trifolium pratense (one capsule per day); GP received placebo (control, one capsule of lactose per day). Treatment lasted 12 months and women were evaluated before and after four, eight and twelve months of treatment by clinical (Kupperman Menopausal Index -KMI and the Golombok Rust Inventory of Sexual Satisfaction) and laboratory exams. At study end, each group had 50 patients. RESULTS: There was a significant decrease of symptoms after four months of treatment according to the KMI, mainly of hot flashes in relation to baseline data for both groups, but not between both groups. Evaluation of sexual satisfaction did not disclose any difference in both groups before and after treatment. CONCLUSION: The 40mg/day Trifolium pratense treatment may not ameliorate menopausal symptoms or improve sexual satisfaction.

Timpanismo espumoso em bovinos leiteiros em pastagens de Trifolium spp. (Leg.Caesalpinoideae) / Leguminous bloat in dairy cattle on Trifolium spp. pastures

O pastejo de plantas leguminosas que provocam fermentação excessiva pode causar surtos de timpanismo e mortes em ruminantes. Em uma propriedade no município de São Francisco de Paula, Rio Grande do Sul, oito bovinos de um total de 66 morreram subitamente, ao haverem sido transferidos de um potreiro de campo nativo, para outro cuja pastagem era composta por Trifolium repens e Trifolium pratense. Os animais foram encontrados mortos no amanhecer do dia seguinte à transferência, não foram observados sinais clínicos prévios. Os principais achados macroscópicos incluíram aumento de volume abdominal, protrusão de vagina e língua, distensão ruminal, fígado de coloração pálida e aumento do baço. Na histologia, havia congestão e edema pulmonares e hiperplasia linfóide difusa e acentuada no baço. A evidência de ingestão das leguminosas associada aos achados patológicos e à ausência de microrganismos no exame bacteriológico confirmaram o diagnóstico de timpanismo.

Leguminous bloat may occur in cattle which graze pastures consisting of lush forages. In a dairy farm located on the municipality of São Francisco de Paula, Rio Grande do Sul, eight out of 66 cows died suddenly after being transferred to a paddock whose pastures were composed of Trifolium repens and Trifolium pratense. Animals were found dead in the morning of the next day after being transferred; no clinical signs were noticed. Main gross findings included enhanced abdominal volume, protrusion and congestion of the tongue and vagina, ruminal distension, pale liver, and enhanced spleen. Histologically, there were lung congestion and edema, and splenic lymphoid hyperplasia. The evidence of leguminous forages consumption associated with the pathological findings and the absence of growth on bacteriology confirmed the diagnosis.

Ultrastructural changes of the egg apparatus associated with fertilisation of natural tetraploid Trifolium pratense L. (Fabaceae)

The aim of this study is to describe the ultrastructural changes of the egg apparatus associated with fertilisation of the natural tetraploid Trifolium pratense. The pollen tube enters one of the synergids through the filliform apparatus from the micropyle. Before the entry of the pollen tube into the embryo sac, one of the synergids begins to degenerate, as indicated by increased electron density and a loss of volume. This cell serves as the site of entry for the pollen tube. Following fertilization, the vacuolar organisation in the zygote changes; in addition to the large micropylar vacuole, there are several small vacuoles of varying size. Ribosomal concentration increases significantly after fertilisation. In T. pratense, ultrastructural changes between the egg cell and zygote stages are noticeable. Several marked changes occur in the egg cell because of fertilisation. The zygote cell contains ribosomes has many mitochondria, plastids, lipids, vacuoles. After fertilization, most of the food reserves are located in the integument in the form of starch. The zygote shows ultrastructural changes when compared to the egg cell and appears to be metabolically active.

Plant regeneration of natural tetraploid Trifolium Hum pratense L

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.