Abstract
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Background: Traditional medicine systems have long harnessed potent toxic botanicals, employing classical processing techniques to render them safe and therapeutically effective. Semecarpus anacardium (SA) exemplifies this duality within the Ayurvedic tradition — a plant of considerable therapeutic relevance whose inherent toxicity in its raw form restricts clinical application. Ayurvedic practitioners have historically employed Shodhana, a classical detoxification process, to overcome this limitation; however, the scientific community has yet to rigorously characterize how different Shodhana protocols differentially modulate the phytochemical and biological profile of SA.
Objective: This study aimed to evaluate the differential impact of three classical Shodhana methods — Coconut Water, Milk, and Cow Urine + Milk processing — on the phytochemical composition and in vitro anticancer activity of S. anacardium relative to its raw, unprocessed form.
Methodology: Authenticated SA nuts underwent three distinct Shodhana protocols followed by standardized Soxhlet ethanol extraction. LC-MS/MS profiling characterized the phytochemical changes each processing method induced. In vitro anticancer efficacy and safety assessment employed MTT-based cytotoxicity and AO/EB dual staining for morphological apoptosis analysis on MCF-7 breast cancer and L929 normal fibroblast cell lines. DCFDA fluorescence imaging further probed the mechanism of cell death by quantifying intracellular reactive oxygen species (ROS) generation.
Results: LC-MS/MS profiling revealed distinct phytochemical fingerprints across all four preparations. The raw extract carried a single dominant compound constituting 79% of the total chromatographic area, while each Shodhana method uniquely diversified this profile — Milk Shodhana produced the most balanced redistribution across multiple co-dominant peaks, whereas Cow Urine + Milk processing shifted the elution profile toward more lipophilic compounds, indicating structural transformation of the primary constituent. In vitro evaluation on MCF-7 cells demonstrated that both raw and Cow Urine + Milk-processed extracts induced dose-dependent apoptotic cell death confirmed by AO/EB staining, and significantly elevated intracellular ROS levels detected by DCFDA fluorescence, pointing to an oxidative stress-mediated anticancer mechanism.
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