Maturity related metabolomic analysis of Balanites aegyptiaca fruits with in vitro and in silico cytotoxicity evaluation.

Balanites aegyptiaca (L.) Delile, a medicinal tree, produces an edible fruit widely recognized in traditional medicine for its antidiabetic and liver-enhancing properties. This study investigated the metabolic changes occurring during fruit maturation using integrated nuclear magnetic resonance (NMR) metabolomics approaches, including 1D H (proton), 2D heteronuclear single quantum coherence (HSQC), and 2D J-resolved NMR spectroscopy. A total of forty-five metabolites were identified and quantified, with key metabolites characterizing each maturation stage. Metabolic profiling indicated that immature fruits were characterized by elevated concentrations of amino acids, alkaloids, and organic acids, while mature fruits predominantly accumulated monosaccharides. Chemometric analyses and hierarchical clustering confirmed a significant metabolic differentiation between immature and mature fruit stages. Pathway analysis identified significant alterations predominantly in starch-sucrose metabolism, pyruvate metabolism, and the citrate cycle during maturation. Cytotoxic evaluation revealed that polar extracts from immature fruits exhibited superior cytotoxic activity against hepatocellular carcinoma cells (IC = 117.7 µg/mL) compared to mature fruit extracts (IC = 270.4 µg/mL). Molecular docking analysis further demonstrated that metabolites upregulated in immature fruits, like theophylline, showed a strong binding affinity (- 5.317 kcal/mol) to the anti-apoptotic protein BCL-2, suggesting their potential role in apoptosis regulation. This study provides insights into the metabolic dynamics during Balanites aegyptiaca fruit maturation, highlighting the superior therapeutic potential and significant cytotoxic activity of immature fruits compared to traditionally utilized mature fruits.