The investigation of phenolic compounds and antioxidant activity of zigzag clover (Trifolium medium L.) plants.

The aim: to assess quantitative composition of phenolic compounds and flavonoids in the raw materials of zigzag clover (Trifolium medium L.) cultivated in Lithuania, as well as to evaluate the antioxidant activity of these plants. The objectives of the study: 1) To optimize the extraction conditions of phenolics compounds from T. medium raw materials. 2) To analyze the quantitative composition of total phenolic and flavonoids and their variation patterns in T. medium raw materials using spectrophotometric methods. 3) To evaluate antioxidant activity and its variability in extracts of T. medium raw materials using DPPH and FIC assays. 4) To estimate correlation between quantitative composition of total phenolic compounds, flavonoids and antioxidant activity. 5) To compare the content of total phenolics, flavonoids and antioxidant activity in raw materials of T. medium and two diploid cultivars of T. pratense. Research methodology: The study was carried out using samples of the raw materials of cultivated zigzag and red (two cultivars: Liepsna and Kiršinai) clovers. The method of extraction - ultrasonic agitation, extractant – 70% (V/V) ethanol, extraction time – 15 min, extraction temperature - 50ºC. Spectrophotometric Folin-Ciocalteu method was used to determine the total content of phenolic compounds and results were expressed as gallic acid equivalent (mg/g). Specific reaction with AlCl3 was used to determine the total content of flavonoids and results were expressed as rutin equivalent (mg/g). Two spectrophotometric assays, namely DPPH and FIC, were applied to determine radical scavenging and chelating activities of plant extracts. Results and conclusions: This study revealed that T. medium raw materials accumulate significant quantities of total phenolic and flovonoids. Their quantitie varies significantly depending on the harvest year, the phenological stage and the morphological plant part. The highest amount of phenolic compounds was observed in T. medium flowers (64,090 ± 0,406 mg/g) of the second years of growth while the lowest amount of phenolic compounds was detected in extracts of stems (8,436 ± 0,163 mg/g). The highest level of flavonoids was observed in raw materials of leaves (24,658 ± 0,527 mg/g) and the lowest amount of flavonoids was observed in extracts of stems (1,370 ± 0,264 mg/g). The highest antioxidant activity was determined in extracts of T. medium flowers using both DPPH radical scavenging (80,35 ± 2,33%) and FIC assays (78,21 ± 0,94%). The strong correlation between quantitative composition of total phenolics and antioxidant activity evaluated by DPPH (0,838) and FIC methods (0,854) was determined in raw materials of T. medium. This research showed that considerably higher levels of total phenolic compounds were found in T. medium raw materials compared to T. pratense. The comparison of antioxidant properties revealed that extracts from T. medium flowers are characterized by the highest values of antiradical activity whereas extracts from raw materials of diploid T. pratense cultivars were found to by superior regarding the chelating ability