Cyclopamine Concentration from Extraction as a Function of pH

Cyclopamine is a steroidal alkaloid common to Veratrum californicum (V. californicum), a plant that grows in abundance in the mountains of Idaho. Cyclopamine inhibits the hedgehog pathway, a mechanism important in developmental biology and cancer treatment. The extraction of cyclopamine from V. californicum biomass by established methods may result in the degradation of the alkaloid into inactive, but closely related compounds. Isolation of cyclopamine in our lab is currently performed via Soxhlet extraction of the V. californicum biomass, with great care taken to maintain the pH of the solution above 7. Studies have shown that lower pH’s degrade cyclopamine. The current investigation will characterize the impact of pH on extraction efficiency of the bioactive cyclopamine alkaloid. Using varying pH in the extraction method, we will determine the necessary hydroxide concentration to maximize the yield of cyclopamine. An important consideration is the possibility of converting the glycosylated alkaloid, cyclopasine, into cyclopamine under acidic conditions. Thus, this investigation will encompass a range of pH’s to identify optimal extraction conditions that maintain the bioactivity of cyclopamine. The method used to quantify alkaloids from the V. californicum extracts is peak integration from high performance liquid chromatography separation, which also enables the analysis of alkaloid ratios to monitor pH effects on deglycosylation. Mass Spectrometry is used to identify the isolated alkaloids