Integrated Extraction Optimization and HPLC-based Quality Evaluation of Thiophenes from Tagetes erecta Roots

Tagetes erecta L. is widely studied for its flower-derived lutein, which is known for promoting eye health. However, its roots contain uniquely thiophenes-absent from the flowers and leaves-which exhibit valuable bioactivity. Our recent study has reported on patent applications related to their efficacy in modulating benign prostatic hyperplasia, underscoring their pharmaceutical and functional potential. Despite this, no optimized extraction or quality evaluation of thiophene-rich T. erecta roots has been reported. This study aimed to establish a reliable extraction strategy for thiophene-rich T. erecta roots and to ensure chemical consistency through validated HPLC quantification and chromatographic fingerprint similarity analysis. Three major thiophenes-5-(4-hydroxybut-1-ynyl)-2,2'-bithiophene (1), 5-(4-acetoxybut-1-ynyl)-2,2'-bithiophene (2) and 5-(3-buten-1-ynyl)-2,2'-bithiophene (3)-were isolated and structurally confirmed. The extraction variables (solvent, plant part, method, sample amount and time) were optimized. Quantification was performed via validated HPLC-PDA using in-house purified standards (≥ 98%), and fingerprint similarity was evaluated. Ultrasonic extraction for 2 h with 5 g of root powder in 95% ethanol gave optimal yield and reproducibility. The HPLC method exhibited excellent linearity (R ² >  0.999), precision (RSD < 1%) and recovery (93.20%-105.24%). Fingerprint analysis of 13 common peaks revealed high similarity (0.984-1.000), indicating stable chemical profiles. This study provides a validated workflow for thiophene-rich T. erecta roots, highlights the significance of root-derived thiophenes and offers a practical basis for process development, quality control and future standardization of thiophenes and related constituents.