Box-Behnken Design Optimization of High-Pressure Processed Bitter Melon (Momordica charantia) Leaf Extract Enhancing...

Bitter Melon Leaf Extract (BMLE) possesses potential anticancer and anti-inflammatory properties; however, conventional extraction methods restrict phytochemical yield and bioactivity. Here, we optimized extraction using High-Pressure Processing (HPP) with Box-Behnken Design (BBD) and Response Surface Methodology (RSM). The optimized extract (O-BMLE) demonstrated significantly higher total flavonoid content (27.7 vs. 8.7 mg RE/g) and FRAP antioxidant capacity (96.5 vs. 71.2 μmol TE/g) compared to conventional BMLE. Additionally, O-BMLE exhibited enhanced cytotoxicity (A549 IC₅₀: 58.7 vs. 147 μg/mL) and selectivity (SI: 5.03 vs. 2.60) against A549, HepG2, and SKOV3 cancer cells while showing minimal effects on 3T3-L1 fibroblasts. In LPS-stimulated RAW264.7 macrophages, O-BMLE selectively inhibited JNK phosphorylation without affecting NF-κB phosphorylation, resulting in suppression of iNOS, COX-2, IL-1β, IL-6, and TNF-α expression as well as nitric oxide production. HPLC analysis revealed equivalent momordicine-I levels (~28 mg/g) between extracts. In contrast, HPLC-qTOF-MS profiling revealed that O-BMLE was enriched in stearidonic acid (66% increase in relative abundance), 4-hydroxybenzoic acid (19.5%), monolinolenin, 6-gingerol, and pedunculoside, which are compounds linked to JNK inhibition, antioxidant activity, and cytokine suppression. These results indicate that HPP-BBD/RSM optimization selectively modifies the bitter melon leaf metabolome, thereby enhancing anticancer and anti-inflammatory activities independently of momordicine-I content alone. O-BMLE may therefore serve as a promising candidate for the development of functional foods and nutraceuticals targeting inflammation-associated cancers.