**Background:** Pomegranate (Punica granatum L.) leaves are a non-edible biowaste generated during fruit processing, with no current industrial applicability despite being a rich source of phenolic compounds, flavonoids, and tannins with known therapeutic properties. This study aimed to evaluate the bioactive properties and phenolic composition of pomegranate leaf hydroethanolic extracts obtained through three different extraction methodologies to support their valorization.
**Methods:** Pomegranate leaves were collected from Quinta do Prado, Vila Flor, Portugal, freeze-dried, and powdered. Three extraction methods were performed using a hydroethanolic solvent (EtOH/H2O, 60:40, v/v) with a solid-liquid ratio of 1:20: microwave-assisted extraction (MAE; 15 min at 80°C, 350 W), ultrasound-assisted extraction (UAE; 5 min at 350 W), and maceration (MAC; 1 h at room temperature with continuous stirring). Phenolic compounds were identified and quantified using HPLC-DAD-ESI/MSn. Bioactive properties assessed included antioxidant activity (TBARS and cellular antioxidant assays), cytotoxicity against four human tumor cell lines (MCF-7, NCI-H460, AGS, CaCo-2) and hepatotoxicity in PLP2 cells, anti-inflammatory activity (NO inhibition in RAW 264.7 cells), skin-beneficial properties (viability in HFF-1 and HaCaT cells, tyrosinase inhibition), and antimicrobial activity against clinical and foodborne pathogens.
**Key Results:** Thirty-three phenolic compounds were tentatively identified, including 13 phenolic acids, 15 flavonoids, and 6 hydrolyzable tannins. MAE extract had the highest total phenolic content (15.9 ± 0.3 mg/g), followed by MAC (10.1 ± 0.1 mg/g) and UAE (6.6 ± 0.1 mg/g). Gallic acid was the most abundant phenolic acid across all extracts (MAE: 0.95 mg/g; UAE: 1.19 mg/g; MAC: 1.45 mg/g). (-)-Epicatechin was the major flavonoid in MAE (2.40 mg/g) and MAC (0.83 mg/g), while quercetin-O-glucuronide dominated in UAE (1.20 mg/g). Granatin B was the most abundant tannin in MAE (3.0 mg/g). For antioxidant activity, MAC and MAE showed the lowest TBARS IC50 values (0.83 and 0.86 μg/mL, respectively), outperforming trolox by 10.7- and 10.9-fold. No cellular ROS inhibition was observed at 2000 μg/mL. All extracts showed cytotoxic effects against tumor cell lines, with AGS (gastric adenocarcinoma) being most sensitive (GI50: 19-26.3 μg/mL). UAE extract showed the highest overall cytotoxicity. Hepatotoxicity was observed in PLP2 cells (GI50: 54-57 μg/mL). No anti-inflammatory activity was detected (IC50 > 400 μg/mL). MAE and MAC extracts inhibited tyrosinase by >30% at 300 μg/mL. All extracts maintained >70% viability in HFF-1 fibroblasts at 400 μg/mL. Antimicrobial testing showed MAE and MAC were most effective, with MRSA being the most sensitive Gram-positive strain (MIC: 0.3-0.6 mg/mL) and Klebsiella pneumoniae the most sensitive Gram-negative (MIC: 0.6 mg/mL). Against food contaminants, Salmonella enterica and Staphylococcus aureus showed highest sensitivity. Antifungal activity was modest, with Aspergillus brasiliensis most sensitive (MIC: 0.6-2.5 mg/mL).
**Clinical Implications:** This study provides experimental evidence that pomegranate leaves, currently discarded as agricultural waste, contain a diverse array of bioactive phenolic compounds with antioxidant, cytotoxic, anti-tyrosinase, and antimicrobial properties. The extracts demonstrated particular promise against gastric and colorectal cancer cell lines and multi-drug resistant bacteria including MRSA. The favorable safety profile on skin cell lines supports potential dermatological applications. These findings support the sustainable valorization of pomegranate leaf biowaste as a functional ingredient for the food, pharmaceutical, and cosmetic industries, though further studies on molecular mechanisms and in vivo efficacy are needed.