Bioactivity Assessment of \u3cem\u3eVeratrum californicum\u3c/em\u3e Alkaloids

The native Idaho plant Veratrum californicum is known to contain steroidal alkaloids that function as inhibitors of hedgehog signaling, a pathway utilized for the growth and differentiation of cells as well as proper tissue development. Veratrum californicum was originally noticed when pregnant ewes consumed the plant and later gave birth to lambs with craniofacial mutations such as a cyclopean eyes. These malformations were caused by the plant’s steroidal alkaloid cyclopamine blocking the hedgehog signaling pathway. This same pathway is used by more than 20 types of cancer for multiplication of cells. Additional alkaloids have been found in Veratrum californicum, which are being studied in order to identify additional hedgehog signaling inhibitors. There are at least seven compounds detected in extracts that are completely uncharacterized, twelve with proposed identities, and the six known alkaloids veratrosine, cycloposine, veratramine, cyclopamine, isorubijervine, and muldamine all within Veratrum californicum. In this thesis these molecules were extracted together from the root and rhizome of the plant, and then separated into five fractions using high performance liquid chromatography. Mass spectrometry analysis identified the presence of twenty five alkaloids (nine more than literature precedent), and software processing of the data predicted the molecular formulas for each known, suspected, and unknown alkaloid. The bioactivity of the raw extract and each fraction was assessed by comparison to 0.1 μM cyclopamine, and it was found that three fractions suppressed hedgehog signaling to a greater extent, indicating the presence of bioactive constituents worthy of further investigation. Five fractions were collected at 2.5 min intervals, beginning at 10.25 min from time of raw extract injection. Fraction 1 was most potent at Hh signal inhibition, and was determined to contain veratrosine, cycloposine, and an isomer of each, followed by fractions 2 and 4, which possessed elevated bioactivity, but to a lesser extent than fraction 1. Fraction 2 was predicted to be comprised of cycloposine, its isomer, an isomer of veratrosine, tetrahydrojervine, veratramine, dihydrojervine, its isomer, etioline, and five unknown compounds. Fraction 4 was composed of cyclopamine, and suspected to contain isomers of veratramine and cyclopamine, isorubijervine, and muldamine, as well as two unknown alkaloids