Optimized two-step flash chromatography method for large-scale isolation of linusorb and its antioxidant capacity evaluation

The study presents a novel purification method tailored for a range of linusorbs with comparable polarity, leveraging flash chromatography, a rapid and convenient technique, for large-scale purification of linusorbs. A two-step approach employing silica-phenylhexyl strategy was first used for linusorb (LO, also known as cyclolinopeptide) preparation, yielding fractions of cyclolinopeptide A, E, O with purity exceeding 90 % for each 850 mg linusorbs load. Additionally, other eight fractions containing various LOs exhibited average purities ranging from 60 % to 83 %. Comparative assessment of antioxidant capacity of individual LOs elucidated the role of specific amino acid residues. Met residues initially contributed to LOs' antioxidant effects but declined due to oxidation of Met to MetO. Meanwhile, Trp residues exhibited stronger antioxidant capacity, enhancing the capacity of LOs lacking Met. Furthermore, the Phe-Phe structure was identified as contributing to the antioxidant effect of linusorbs. This study not only provides an efficient and scalable method for the purification of LOs but also offers insights into their antioxidant mechanisms.