Abstract
BACKGROUND: Withania somnifera (L.) Dunal, commonly known as ashwagandha, is a cornerstone of Ayurvedic medicine and has demonstrated anti-metastatic properties, including the ability to mitigate the cytotoxic effects of carcinogens and chemotherapeutic agents. Neuroblastoma (NB), a highly aggressive paediatric cancer, accounts for approximately 15% of childhood cancer-related deaths. Despite intensive treatment, over 50% of NB cases experience tumor recurrence and debilitating long-term effects. This study aimed to evaluate the anti-progression effects of W. somnifera root fractions on the human NB Kelly cell line at sub-cytotoxic concentrations and to identify the active bioactive constituents. METHODS: W. somnifera roots were extracted using 95% ethanol and subsequently fractionated via vacuum liquid chromatography with a methanol-water gradient elution, yielding twelve fractions. Kelly cells were treated with each fraction at sub-cytotoxic concentrations, as determined by MTT assay. Treated cells were then subjected to transwell extracellular matrix invasion and fibronectin adhesion assays. Data were analysed using one-way ANOVA (GraphPad Prism), with statistical significance set at P ≤ 0.05. Bioactive fractions were further subfractionated by preparative HPLC, and major constituents were tentatively identified using GC-MS. RESULTS: Fraction 9 (eluted with 70% methanol) exhibited the highest anti-invasive activity, whereas Fraction 10 (eluted with 80% methanol) demonstrated the most potent and statistically significant (P = 0.0409) anti-adhesive effect compared to vehicle-treated cells (0.5% DMSO). Subfraction analysis revealed that Subfraction 10/1 had a significant anti-adhesive effect (P = 0.0482), while subfractions 10/3 and 9/2 showed non-significant anti-adhesive effects. GC-MS analysis of subfractions 9/2, 10/1, and 10/3 revealed the presence of four previously unreported compounds in W. somnifera. CONCLUSIONS: Constituents of W. somnifera roots exhibit promising anti-metastatic activity against neuroblastoma cells, highlighting their potential to complement existing chemotherapeutic regimens and reduce associated long-term side effects. CLINICAL TRIAL NUMBER: Not applicable.