Clovamide and Flavonoids from Leaves of Trifolium pratense and T. pratense subsp. nivale Grown in Italy

The phenolic content and composition in leaves of Trifolium pratense (red clover) and T. pratense subsp. nivale (snow clover) grown in Italy were evaluated by means of ultraperformance liquid chromatography (UPLC) coupled with photodiode array and mass spectrometry detectors. Compound identification was based on UV and MS data comparing results with those of reference compounds. Quantitative evaluation of all detected compounds was based on calibration curves obtained with available standards. Several phenolics were identified in both extracts, including clovamide, flavonols and isoflavones as their glycosilated and malonated derivatives. The total phenolic content was higher in red clover (53.7 ± 2.2 mg/g dry weight) than in snow clover (44.4 ± 4.9 mg/g dry weight). Red clover contained higher amounts of clovamide and isoflavones (15.6 ± 0.6 and 24.6 ± 1.6 mg/g dry weight, respectively) than snow clover (8.2 ± 0.1 mg/g and 16.9 ± 0.4 mg/g dry weight, respectively), while flavonols were quantified almost in the same amount in both extracts (13.2 ± 0.6 mg/g and 15.8 ± 0.6 mg/g dry weight in red clover and snow clover, respectively). Red clover was characterized by the presence of quercetin, formononetin and biochanin A derivatives as the most abundant flavonoids, whereas snow clover was characterized by higher amounts of quercetin and prunetin derivatives. This investigation, conducted for the first time on phenolics from T. pratense subsp. nivale, revealed the presence in this plant of several flavonoid derivatives the same as in T. pratense. The higher amount of prunetin in snow clover suggest a possible role of this isoflavone as a chemotaxonomic marker for this subspecies. Moreover, snow clover may represent an interesting new source of natural isoflavones with a different concentration pattern than in red clover.