Remediation of pyrrolizidine alkaloids in plant extracts: a continuous liquid-liquid chromatography approach for red clover

Red Clover ( Trifolium pratense ) is widely acknowledged in traditional medicine for its therapeutic potential in managing osteoporosis, cardiovascular diseases, arthritis, eczema, asthma, and other conditions. Its beneficial effects are primarily attributed to the rich content of isoflavones, which exhibit estrogenic and antioxidant properties. Pyrrolizidine alkaloids (PAs) are toxic secondary metabolites with hepatotoxic and carcinogenic properties. Red Clover extracts may become contaminated with PA-containing weeds upon harvesting and processing. PA remediation is crucial for safety and therapeutic efficacy; however, efficiently reducing PA levels while preserving isoflavones remains a significant challenge, especially on an industrial scale [ 1 ]. Centrifugal Partition Chromatography (CPC) is a liquid-liquid chromatographic technique that separates solutes based on their differential partitioning between two immiscible phases. Compared to batch processing, continuous CPC can enhance productivity and scalability. Continuous operation in CPC is enabled by Multiple Dual Mode (MDM), where periodic shifts between ascending and descending modes alternate the roles of the mobile and stationary phases in each cycle [ 2 ]. This study evaluated the effectiveness of CPC in continuous manufacturing mode for the removal of PAs from crude Red Clover extracts while preserving isoflavone content. Additionally, its performance was compared to conventional batch processing. Results demonstrated that alkaloid content was reduced by over an order of magnitude, achieving regulatory compliance (