| Summary: | Parthenolide, a strong cytotoxic compound found in different parts of <i>Tarchonanthus camphoratus</i> which motivated the authors to develop an optimized microwave-assisted extraction (MEA) method using Box–Behnken design (BBD) for efficient extraction of parthenolide from the stem of <i>T. camphoratus</i> and its validation by high-performance thin-layer chromatography (HPTLC) and cytotoxic analysis. The optimized parameters for microwave extraction were determined as: 51.5 °C extraction temperature, 50.8 min extraction time, and 211 W microwave power. A quadratic polynomial model was found the most suitable model with <i>R</i><sup>2</sup> of 0.9989 and coefficient of variation (CV) of 0.2898%. The high values of adjusted <i>R</i><sup>2</sup> (0.9974), predicted <i>R</i><sup>2</sup> (0.9945), and signal-to-noise ratio (74.23) indicated a good correlation and adequate signal, respectively. HPTLC analyzed the parthenolide (R<sub>f</sub> = 0.16) content in <i>T. camphoratus</i> methanol extract (TCME) at λ<sub>max</sub> = 575 nm and found it as 0.9273% ± 0.0487% <i>w</i>/<i>w</i>, which was a higher than expected yield (0.9157% <i>w</i>/<i>w</i>). The TCME exhibited good cytotoxicity against HepG2 and MCF-7 cell lines (IC<sub>50</sub> = 30.87 and 35.41 µg/mL, respectively), which further supported our findings of high parthenolide content in TCME. This optimized MAE method can be further applied to efficiently extract parthenolide from marketed herbal supplements containing different <i>Tarconanthus</i> species.
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