Cytotoxic Potential Evaluation of Innovative Pressurised Cyclic Solid–Liquid Extracts from Withania somnifera

Ethnopharmacological relevance. Withania somnifera (L.) Dunal, widely used in traditional medical systems such as Ayurveda, Unani, and Middle Eastern folk medicine, is valued for its adaptogenic, anti-inflammatory, neuroprotective, antimicrobial, and anticancer properties. These activities are primarily attributed to withanolides, with Withaferin A recognized as one of the most bioactive constituents. Although traditional preparations often rely on the root, leaf use provides a more sustainable alternative and may yield significant quantities of active metabolites. Identifying efficient, modern extraction technologies that can enhance the recovery of bioactive compounds from leaves is essential for developing effective, standardized ethnopharmacological formulations. Materials and methods. Plants of W. somnifera grown from seeds were subjected to different environmental conditions (control, drought, cold, yeast extract treatment). Leaves were extracted using Pressurized Cyclic Solid–Liquid Extraction (PCSLE) with hydroalcoholic solvents and compared with conventional infusion of dried leaves. Extracts were fractionated with solvents of varying polarity and analyzed by TLC, HPLC, and NMR for quantification of Withaferin A. Expression levels of key withanolide-biosynthetic genes (CAS, SMT1, DWARF1, CYP71, CYP76) were assessed using qRT-PCR. Antimicrobial activity of pure Withaferin A, aqueous extract, and hydroalcoholic PCSLE extract was evaluated through MIC and MBC assays against Gram-positive and Gram-negative strains. Cytotoxic activity was measured via MTT assays in six human cancer cell lines after 3, 6, and 24 h of treatment. Results. PCSLE yielded substantially higher levels of Withaferin A than traditional infusion, especially in medium-polarity fractions (chloroform and ethyl acetate), with concentrations reaching 0.70% in fresh leaf mass (4.8% dry weight), compared to 0.11% obtained by infusion. Gene expression analysis revealed that 24-week-old plants exhibited the highest transcription of withanolide-biosynthetic genes, and drought stress significantly upregulated CAS, SMT1, DWARF1, CYP71, and CYP716, indicating enhanced metabolic flux toward withanolide production. Hydroalcoholic PCSLE extracts showed broad-spectrum antimicrobial activity, with MIC and MBC values comparable to pure Withaferin A and demonstrating bactericidal effects against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes. The aqueous extract showed activity only against Gram-positive strains. Cytotoxicity assays demonstrated an optimistic, dose-dependent reduction in cell viability across all tumour cell lines treated with the hydroalcoholic PCSLE extract, closely mirroring the activity of pure Withaferin A and consistently exceeding the effect of the aqueous extract. IC50 values confirmed the high bioactive content of PCSLE extracts and suggested mechanisms like those known for Withaferin A. Conclusions. PCSLE proved to be a highly efficient extraction technology for obtaining leaf extracts rich in Withaferin A, outperforming conventional extraction methods while exploiting sustainable plant tissue. Developmental stage and drought stress significantly modulated the expression of genes involved in withanolide biosynthesis, highlighting agronomic strategies capable of enhancing metabolite production. Hydroalcoholic PCSLE extracts exhibited antimicrobial and cytotoxic activities comparable to pure Withaferin A, supporting their relevance as promising therapeutic candidates. These findings advocate for the use of W. somnifera leaves as a sustainable source of bioactive compounds and demonstrate that advanced extraction technologies can contribute to the development of innovative ethnopharmacological preparations for antimicrobial and anticancer applications.