Influence of green aqueous extraction techniques on selected phytochemical markers, biological screening responses, and acute oral toxicity of Cannabis sativa L. extracts

Cannabis sativa L. is an industrial and medicinal plant recognized as a source of bioactive metabolites with potential pharmaceutical applications. This study compared four water-based extraction protocols—infusion, Soxhlet extraction, ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE)—applied to extracts obtained from female cultivated C. sativa L. plants. Infusion yielded the highest apparent total phenolic, flavonoid, and flavonol contents (72.38 ± 1.01 mg GAE g −1 , 15.05 ± 0.07 mg QE g −1 , and 16.69 ± 0.82 mg QE g −1 , respectively), whereas UAE showed higher chlorophyll a and chlorophyll b contents. FTIR and 1 H/ 13 C NMR revealed comparative fingerprints, while targeted LC-MS/MS quantified selected phenolic compounds and cannabinoids, with UAE showing higher cannflavin B and Δ9-THC concentrations and infusion showing the highest measured isorhamnetin, CBDA, and total CBD concentrations within the targeted panel. Radical-scavenging responses differed between assays: infusion showed the lowest ABTS IC 50 value (0.72 ± 0.003 mg mL −1 ), whereas UAE showed the lowest DPPH IC 50 value (0.20 ± 0.01 mg mL −1 ). All extracts inhibited Lepidium sativum root elongation, indicating preliminary phytotoxic activity. No acute lethality or treatment-related clinical signs were observed in Swiss albino mice at the tested limit dose of 2000 mg kg −1 ; however, extract-dependent biochemical and hematological changes were detected. Under the tested, non-normalized operating conditions, the extraction protocols produced distinct responses across colorimetric indices, selected LC-MS/MS analytes, and preliminary biological screening assays. Further standardized chemical characterization and extended toxicological evaluation are required.