Connecting chemical and biological properties to identify new functional materials: A study on Trifolium nigrescens extracts

In this study, extracts of the plant species Trifolium nigrescens collected in Turkey were examined. The extracts were obtained by maceration as a conventional method for the extraction of bioactive metabolites, using solvents of different polarity (ethyl acetate, ethanol, 70 % ethanol and water). The antioxidant, enzyme inhibitory and anti-biofilm potential was investigated using in vitro assays, as well as chemical profiles. Ellagic acid was the dominant phenolic acid (4115.87 mg/kg), delphinidin-3-galactoside was the most abundant anthocyanin (24789.25 mg/kg), while hyperoside as flavonoid was identified in the highest concentration (42450.94 mg/kg). Ethanol and 70 % ethanol were the most effective extracts in the radical scavenging and reducing power assays. However, the metal chelating abilities of the ethyl acetate (14.80 mg EDTAE/g) and 70 % ethanol (14.13 mg EDTAE/g) extracts were close to each other. In terms of AChE and tyrosinase inhibition, 70 % ethanol was the most active with these values of 2.34 mg GALAE/g and 48.97 mg KAE/g, respectively. Based on the microbiological aspects, the extracts were able to inhibit the mature biofilm or act on the metabolism of their sessile cells, with the percentage of inhibition reaching almost 60 %. In some cases, such as in the evaluation of anti-biofilm activity against Acinetobacter baumannii, the presence of water in the extract buffer had a positive effect on the inhibition of mature biofilm. Furthermore, molecular docking and 100-ns molecular dynamics simulations were conducted to assess the interaction of selected compounds with both microbial proteins and all five standard enzymes. The correlation analysis identified some polyphenols, including phenolic acids and flavonoids, which were most involved in enhancing or attenuating the antibiofilm effect of the extracts.