Red clover (Trifolium pratense L.) is a perennial legume is a rich source of isoflavones, which are reported to have beneficial health effects [1]. Moreover, it contains significant amounts of phenolic compounds which are known for their potential bio-active antioxidant properties and radical scavenging capacity [2].
The aim of this study was to investigate the antioxidant activity of Trifolium pratense L. blossoms extracted using different extraction methods.
Ultrasound assisted extraction was performed using $1 \pm 0.001$ g of dried and milled flower heads and 30 mL of 50% ethanol. Temperature during extraction – 40 and 60 °C. Ultrasound power 250 W and extraction time 10 and 30 min [3]. Thermal processing was performed after sonication placing extract in a heating mantle under a reflux condenser for one hour. Heat-reflux extraction was done using $1 \pm 0.001$ g of dried and milled flower that was mixed with 30 mL of 50% ethanol in a 250 mL round bottom flask and refluxed in the sand bath at 100 °C for 1 hour. Maceration [4] results was used as control. The antioxidant activity of extracts has been evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) [5] and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) [6] radical scavenging methods. All experiments were performed in triplicate.
Spectrophotometric analysis showed that the antioxidant activity differed significantly between differently prepared extracts. Highest radical scavenging capacity in both antioxidant activity methods were determined in sample UT4 (88.23% and 40.29% ABTS and DPPH results respectively), that was prepared using ultrasound (30 min; 60 °C). Using ultrasound, the increase in antioxidant activity was observed by extending the duration of the ultrasonic treatment from 10 to 30 minutes. The lowest antioxidant activity was found in M sample, that was prepared using maceration and used as control (57.82% and 13.59% ABTS and DPPH results respectively). Comparing ABTS and DPPH methods, DPPH showed almost 2-3 times lower antioxidant activity in extracts, but DPPH is a more stable radical and the results of the extracts are more repetitive.