Stability of protoanemonin in plant extracts from Helleborus niger L. and Pulsatilla vulgaris Mill

The concentration levels and stability of protoanemonin, a characteristic constituent of Ranunculaceae species with antimicrobial and fungicidal properties, were studied for the first time in plant extracts prepared from Helleborus niger L. and Pulsatilla vulgaris Mill. using fermentative production processes. Protoanemonin levels quantified by HPLC-DAD analysis were 0.0345 and 0.0662 mg/g in two freshly prepared Helleborus (whole, flowering plant) extracts and 0.3875 mg/g and 0.4193 mg/g in Pulsatilla (flowers) extracts. Protoanemonin proved to be rather instable in aqueous-fermented extracts stored at 15 °C in the dark, and its concentration decreased rapidly over 12 months of storage independently of the plant species. The decrease was most pronounced when initial concentrations were high (decrease by about 70%). In contrast, low protoanemonin levels remained stable in solution for more than 12 months. Anemonin, the dimer of protoanemonin, was detected in increasing concentrations only in Pulsatilla samples, but its concentration only accounted for less than 50% of the theoretically expected amount. With respect to fermented extracts, both physical processes such as self-polymerization and adsorption/binding to other extract constituents as well as biodegradation were concluded to be responsible for protoanemonin decline. As opposed to plant extracts, both protoanemonin and anemonin levels decreased in 0.22 μm-filtered samples stored in vials. This may be explained by a reduced release from plant material in combination with physicochemically induced degradation. Reduction was most pronounced upon light exposure and elevated temperatures, clearly indicating that photochemical degradation is involved. Contents of protoanemonin in a set of extract batches were 0.0896 ± 0.0125 mg/g and 0.0618 ± 0.0180 mg/g in Helleborus and Pulsatilla extracts, and anemonin levels were 0.0230 ± 0.0076 mg/g and 0.0482 ± 0.0282 mg/g, respectively. Due to its antibiotic effects, but also its reactivity, protoanemonin is a therapeutically and toxicologically relevant constituent, and its concentration should therefore be carefully monitored.

Keywords: Antibiotic; Black hellebore; Fermented extracts; HPLC-DAD; Pasque flower; Validation.