Integrated Metabolomics, Network Pharmacology, and Cell-Based Validation of Indigenous Rice Bran Cultivars against Colorectal Cancer Chemoprevention

In Ayurveda, indigenous rice cultivars are highly regarded, with pigmented landraces described as Rasāyana formulations known for rejuvenating properties. Despite their ethnopharmacological significance, molecular mechanisms underlying their role in colorectal cancer (CRC) prevention remain poorly understood. This study employed an integrated approach combining metabolomics, network pharmacology, and cell-based validation to explore the CRC chemopreventive potential of indigenous rice bran cultivars.

Rice bran samples from seven pigmented and two non-pigmented cultivars were sequentially extracted. Methanolic extracts demonstrated superior phytochemical enrichment and antioxidant activity, evidenced by FRAP, DPPH, and ABTS assays, with pigmented cultivars exhibiting higher bioactivity. Initial untargeted LC–MS/QTOF metabolomics identified 25,000 features, refined to 1,554 reproducible metabolites. Multivariate analyses separated pigmented from non-pigmented cultivars, identifying 146 significantly altered metabolites and 17 putative rice bran biomarkers.

Network pharmacology identified seven bioavailable metabolites sharing 32 common CRC targets. Protein-protein interaction and pathway enrichment analyses identified hub genes including EGFR, PIK3CA, PIK3CB, KDR, ESR1, AR, and MET with enrichment in the PI3K-Akt signaling pathway, suggesting mechanisms related to oxidative stress, apoptosis, and tumor cell proliferation.

These predictions were validated using MTT assays at 24, 48, and 72 h against HCT116 and HT29 CRC cell lines and non-cancerous HaCaT keratinocytes. Several cultivars exhibited cytotoxicity towards CRC cells in a time- and dose-dependent manner, while demonstrating lower toxicity towards HaCaT cells. One pigmented accession emerged as the most promising candidate, with a 72-hour IC₅₀ of 117.18 µg/mL against HCT116 cells and exceeding 1000 µg/mL in HaCaT cells, resulting in a selectivity index above 8.5.

Collectively, these findings provide experimental and systems-level evidence supporting the Rasāyana relevance of indigenous pigmented rice bran and underscore their potential as sources of chemopreventive metabolites targeting PI3K–Akt-associated CRC pathways. This integration establishes a foundation for discovering functional food-derived anti-cancer bioactives.