Optimizing polyphenol extraction and UPLC-MS/MS analysis from red clover (Trifolium pratense L.) species using response surface methodology

• Optimization study evaluating solvent composition, temperature and time using RSM. • 80% ethanol for 45 minutes at 40°C was optimal for polyphenol extraction. • A TPC of 14.85 mg GAE/g DW was achieved from red clover. • Mild extraction conditions for maximum yield encourages sustainable processing. Polyphenols were extracted from red clover ( Trifolium pratense L.) plants following conventional extraction. The combined factors of ethanol concentration (60–80%), extraction time (15–45 min) and extraction temperature (40–80°C) were evaluated. Central composite design software was used to conduct Response Surface Methodology (RSM) modelling and optimize the polyphenol yield from the red clover. Total phenolic content, antioxidant activity and ultra-performance liquid chromatography mass-spectrometry (UPLC-MS/MS) analysis were used to quantify polyphenols. The RSM model identified ethanolic composition having greatest influence ( p < 0.05) on the polyphenol yield with extraction yields increasing with percentage ethanol. The optimal extraction conditions were identified as 80% ethanol for 45 minutes at 40°C. When compared with an exhaustive polyphenol extraction method (70% ethanol for 21 h at room temperature), the optimal extraction resulted in a 25.6% increase in TPC yield. UPLC-MS/MS was performed to quantify the individual polyphenols present within the sample, in which biochanin A and formononetin were present in abundance. The outcomes of this modelling and optimization study demonstrate that polyphenols from red clover can be extracted efficiently with minimal environmental and energy cost and thus had the capability to serve as a source of valuable nutraceutical and pharmaceutical products.