Breast and prostate cancers contribute substantially to cancer-related morbidity and mortality. Despite significant advancements in cancer treatment modalities, there remains a need to deepen mechanistic understanding and develop improved therapeutic strategies, given toxicity, resistance, and limited selectivity. Natural products have played a pivotal role in drug discovery. The structural diversity of natural compounds allows them to target multiple pathways, making them promising candidates in anticancer drug development. Wrightia tinctoria (WT), widely used in Ayurvedic and traditional medicine, has emerged as a plant of significant pharmacological interest. The present study focuses on identifying the bioactive constituents of WT bark through bioassay-guided fractionation, chromatographic techniques, and mass spectrometric profiling. In the present study, polarity-based fractions, including petroleum ether, hexane, chloroform, ethyl acetate, and methanol, were screened for their antiproliferative effects against MDA-MB-231, MCF-7, and PC-3 cancer cell lines. Among these, the petroleum ether fraction (PFWT) showed potent cytotoxic activity, with IC50 values of 59.73 ± 2.90, 97.06 ± 4.76, and 43.63 ± 2.68 μg/mL, respectively. The gas chromatography–tandem mass spectrometry analysis of sub-fractions from PFWT further tentatively identified 42 chemical compounds, out of which 27 compounds are reported for the first time. Overall, the study demonstrates that PFWT possesses strong and broad antiproliferative activity, reinforcing the value of integrating traditional medicinal knowledge with modern analytical and pharmacological approaches. These findings position WT as a promising and sustainable source of novel anticancer agents and warrant further in-depth investigation.

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