Phenolic Constituents Drive Antimicrobial and Antibiotic-Enhancing Activities of Cannabis sativa Seed Extracts Obtained by Two Extraction Methods

Hemp seeds (<i>Cannabis sativa</i> L.) are a rich source of phenolic compounds with antioxidant and antimicrobial potential. Still, their genotype-dependent variability and ability to enhance antibiotic efficacy remain insufficiently explored. This study compared three Romanian hemp seed cultivars (Lovrin 110, Silvana, and LV 585) extracted by conventional hydroalcoholic extraction (CE) and ultrasound-assisted extraction (UAE) to evaluate their phenolic composition, antimicrobial effects, and synergistic interactions with amoxicillin and miconazole. HPLC identified genotype- and method-dependent differences, with UAE extracts showing substantially higher levels of epicatechin, quercetin, rosmarinic acid, resveratrol, and ferulic acid. These patterns showed stronger antimicrobial inhibition against Gram-positive and Gram-negative bacteria and yeasts, confirmed by MIC, fold-reduction, and percent enhancement assays. The most pronounced synergy occurred in <i>Streptococcus pyogenes</i>, <i>Staphylococcus aureus</i>, <i>Bacillus cereus</i>, and <i>Candida albicans</i>. PCA revealed two dominant phenolic-activity axes: a rosmarinic/resveratrol/ferulic axis associated with potent inhibition in <i>Escherichia coli</i> and <i>C. albicans</i>, and a quercetin-driven axis linked to Gram-positive bacteria. Overall, UAE extracts displayed superior phenolic enrichment and bioactivity, demonstrating that specific phenolic structures-not total phenolic content-govern antimicrobial performance and antibiotic-enhancing potential in hemp seed extracts.