This paper aims to evaluate the influence of preparation and extraction methods on the phytochemical profile and microbiological activity of 12 medicinal plants from the west side of Romania. First, the proximate composition (humidity, proteins, lipids, and ash) and elemental composition of the raw material were evaluated. Two levels of plant shredding were used: coarse shredding (CS) and fine grinding of plants to obtain powder (FG), and three extraction methods: conventional solvent extraction (CES), ultrasound-assisted extraction (UAE), and microwave extraction (MWE). The phytochemical profile investigated referred to antioxidant activity (AA), total polyphenol content (TPC), and flavonoid content (TFC), using spectrophotometric methods, and individual polyphenols detected using the LC/MS method. The preparation/extraction method for each medicinal plant was optimized using statistical analysis. The optimized extracts for each medicinal plant were tested to evaluate the antimicrobial potential against 9 standard strains. The results showed that the sample preparation method before extraction (shredding or grinding) influences the content of phytocompounds by increasing them in powder form. The use of green technologies, especially MWE, leads to the highest content of TPC, TFC, and AA. The TPC value ranged between 4.83–19.2 mgGAE/g DM in the samples CS and between 19.00–52.85 mgGAE/g DM in the samples FG. The highest antioxidant value was found in the Origanum vulgare extract, both in the case of the crushed sample (22.66 mM Fe2+/100 g) and the powder sample (81.36 mM Fe2+/100 g), followed by Melissa officinalis, The TFC values varied in the range of 1.02–3.46 mgQE/g DM when CES was used, between 2.36–12.09 mgQE/g DM for UAE, and between 1.22–9.63 mgQE/g DM in the case of the MWE procedure. The antimicrobial activity highlighted the effectiveness of the extracts, especially on the strains of H. influenzae, C. albicans, C. parapsilosis, S. aureus, and S. flexneri. Reduced antimicrobial activity was recorded for the strains of S. pyogenes and P. aeruginosa. The best antimicrobial activity was registered by Thymus serpyllum, with an inhibition rate of 132.93% against E. coli and 78.40% against C. albicans.
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