Optimization of Cyclotide extraction from Taraxacum officinale flowers

Abstract

A non-destructive protocol was created for extracting, isolating and detecting cyclotides from cultivated T. Officinale flower heads. Optimal extraction was achieved by maceration for 15 minutes in 50% MeOH and steeping plant material at 70 °C for 3 hours. Size exclusion chromatography was applied successfully using a stationary phase with a molecular cut off at 1000-5000 Da yielding a good separation at 280nm. A molar attenuation threshold was calculated from a protein standard with purified Kalata B1 and used to validate isolated fractions. An amber colored fraction containing 0.39 mM protein was applied to a 400 MHz NMR to determine the presence of cyclotides using extraordinary chemical shifts. Kalata B1 was not confirmed but NMR showed fingerprint similarities to the standard and a signal at -0.1 ppm. This work demonstrates the viability of the protocol for future use.