Anti-inflammatory activity and chemical analysis of extracts from Trifolium riograndense

Abstract Aiming to investigate new therapeutic agents with fewer side effects, the number of studies about natural products has increased. Phenolic compounds comprise a well-studied class of abundant plant-derived compounds, whose anti-inflammatory activity has been described. Isoflavones are phenolic compounds that occur mainly in the Leguminosae family, and can be found in many species, such as Trifolium riograndense Burkart, Leguminosae (clover). In this study an HPLC method was used to determine and quantify four isoflavones (genistein, daidzein, formononetin, and biochanin A) in hydrolyzed leaf, flower, stolon, and root extracts of T. riograndense. In vivo anti-inflammatory activity was investigated using the rat paw edema method and in vitro chemotaxis model with a dry extract from the leaves, which had the highest amount of isoflavones. The major isoflavone found in all parts of the plant was formononetin. The chemotaxis assay revealed that the different concentrations (0.2-50 µg/ml) of the dry extract significantly inhibited neutrophil migration in a concentration-dependent manner (more than 90%). In the rat paw edema test, oral administration of clover extract 100 mg/kg was able to significantly inhibit the edema formation induced by carrageenan. In conclusion, chemical analyses showed that Trifolium riograndense is a plant rich in isoflavones and a new interesting option as isoflavone source. The results of the biological tests taken together show that the extract of T. riograndense has anti-inflammatory effect in rodents.

Genetic variability of isoflavones in the USDA red clover core collection

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.

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.