Nutraceutical Characterization, 3D Airway Tissue Metabolic Viability, and Cytotoxic Activity of Melissa officinalis Aqueous Leaf Extract in A549 Lung Adenocarcinoma Cells

Background and Objectives: Melissa officinalis (lemon balm) contains phenolic constituents with antioxidant and cytotoxic activities; however, evidence regarding aqueous leaf extracts in lung adenocarcinoma models and reconstructed human airway tissues remains limited. The novelty of the present study lies in the integrated evaluation of the same aqueous extract through phytochemical and antioxidant characterization, a three-dimensional reconstructed tracheobronchial tissue model, and complementary short- and long-term biological endpoints in A549 lung adenocarcinoma cells. Materials and Methods: Antioxidant activity was evaluated using the DPPH assay, total phenolic content (TPC) using the Folin–Ciocalteu method, and the targeted phenolic profile using UHPLC–MS/MS. EpiAirwayTM tissues were exposed to 1000 µg/mL extract for 24 h, and tissue metabolic viability was assessed using the MTT assay. A549 cells were treated with 20–1000 µg/mL extract for 24 h and evaluated using complementary metabolic, lysosomal, mitochondrial, morphological, membrane-integrity, and clonogenic endpoints. Results: The extract exhibited concentration-dependent DPPH radical-scavenging activity, with an estimated IC50 of 1.37 mg/mL (95% CI: 1.00–1.88 mg/mL), and a TPC of 10.84 ± 0.03 mg GAE/g dry extract. The most abundant targeted analytes were the unresolved 3,5-dihydroxybenzoic acid/3,4-dihydroxybenzoic acid group (2.09 ± 0.60 µg/mL) and the p-coumaric acid/trans-p-coumaric acid group (1.31 ± 0.04 µg/mL). EpiAirwayTM tissue viability was 85.87% following exposure to 1000 µg/mL. In A549 cells, metabolic viability decreased to approximately 59% and 53% at 500 and 1000 µg/mL, respectively, whereas neutral red uptake declined to approximately 50% at 1000 µg/mL. Treatment was also associated with mitochondrial membrane depolarization, concentration-dependent cellular and nuclear alterations, apoptotic-like and necrotic staining patterns, and a reduction in clonogenic capacity to approximately 21% at 1000 µg/mL. Conclusions: The aqueous M. officinalis leaf extract exhibited measurable antioxidant activity, did not cause a marked acute reduction in EpiAirwayTM tissue metabolic viability under the single exposure condition tested, and produced concentration-dependent cytotoxic and sustained antiproliferative effects in A549 cells. These exploratory findings do not establish comprehensive respiratory safety, a specific cell-death mechanism, or tumor selectivity. Nevertheless, the integrated experimental approach supports further investigation using standardized multiple extract batches, matched malignant and non-malignant lung epithelial models, and quantitative molecular endpoints.