Revealing the Anti-Breast Cancer Potential of Essential Oil from Jungga Orange Peel (Citrus jambhiri): Network Pharmacology and In Vitro Studies

Abstract

Essential oils from citrus peels, particularly Citrus jambhiri (Jungga orange), are gaining attention for their anticancer properties due to the presence of monoterpenes like D-limonene, γ-terpinene, and β-pinene. Despite its bioactive profile, CEO remains underexplored. This study evaluates the anticancer activity of Citrus jambhiri essential oil (CEO) using GC-MS profiling, network pharmacology, and in vitro testing on T47D breast cancer cells. CEO was extracted via microwave-assisted hydrodistillation, and its constituents were identified through GC-MS. Target genes were predicted using SwissTargetPrediction, PubChem, and CTD, while breast cancer-associated genes were retrieved from GeneCards and Open Targets. Protein-protein interaction and hub genes were analysed using STRING and Cytoscape, with pathway enrichment assessed using DAVID and ShinyGO. Bioactivity was evaluated via MTT, Annexin V/PI, cell cycle, ROS, and protein expression assays targeting PI3K/Akt/mTOR and BTK pathways. CEO was found to be rich in hydrocarbon monoterpenes, mainly D-limonene (35.68%), γ-terpinene (16.14%), and β-pinene (11.84%). Network pharmacology revealed 406 overlapping targets enriched in PI3K/Akt/mTOR and BTK signalling. Key hub genes included AKT1, EGFR, BTK, and TP53. CEO exhibited moderate cytotoxicity (IC50 = 68 µg/mL) with high selectivity (SI = 4.67) compared to doxorubicin. Cell-cycle analysis showed S-phase arrest and sub-G1 elevation, while Annexin V/PI confirmed apoptotic activity with minimal necrosis. ROS levels remained unchanged, and CEO significantly downregulated PI3K, mTOR, and BTK, sparing Akt. CEO demonstrates selective anticancer activity through S-phase arrest, non-oxidative apoptosis, and inhibition of PI3K/mTOR-BTK signalling in T47D cells, suggesting its promise as an adjuvant in breast cancer therapy alongside conventional drugs such as doxorubicin.