Hedgehog antagonists cyclopamine and dihydroveratramine can be mistaken for each other in Veratrum album

Abstract

A toxic plant, Veratrum album (ssp. viriscens), was found to have an inhibitory effect on Hedgehog (Hh), a developmental signaling pathway that has been shown to be active during development, in adult stem cells and in numerous human tumors. Based on earlier studies it was believed that the known Hh inhibitor cyclopamine was present in V. album (ssp. viriscens). Here we show that instead of cyclopamine, dihydroveratramine (DHV) was found in V. album (ssp. viriscens). These compounds are easily mistaken for each other, as both substances share the same molecular weight, and the same main MS/MS fragments. DHV was found to be a less potent Hh inhibitor compared to cyclopamine. This is the first reported occurrence of DVH in nature.

Native <em>V. Californicum</em> Alkaloid Combinations Induce Differential Inhibition of Sonic Hedgehog Signaling<strong> </strong>

“…Peak 4, observed in only in the root/rhizome extract has an m/z and predicted molecular formula consistent with etioline. In this study, potential cyclopamine isomers (see Figure 1g) were observed in the root/rhizome extracted analyzed by LC-MS. One of these potential cyclopamine isomers may be dihydroveratramine, which has previously been identified in Veratrum album by Wilson, et al [31]. However, the relative retention time between dihydroveratramine and cyclopamine observed by Wilson, et al does not support this conclusion, because dihydroveratramine (RT: 13.66 min) was observed to elute prior to cyclopamine (RT: 15.09 min), whereas the purported cyclopamine isomer observed in this study elutes after cyclopamine (see Table 1) under similar HPLC conditions.…”

“…The molecular formula and mass of Peak 1 is consistent with that expected for glycosylated isorubijervine or etioline. Glycosylate etioline has previously been reported to be present in the root of Solanum spirale [31]]. Additional investigation would be required to determine this definitely.…”

Identification and characterization of in vitro and in vivo metabolites of steroidal alkaloid veratramine

“…Veratramine (Figure 1b), a secondary amine of veratrum steroidal alkaloids and the major bioactive constituent in Veratrum nigrum L., was first isolated by Saito in 1940 and verified by Gaffield in 1986 [4,5]. Since then, it has been used as a marker compound for the quality control of Veratrum nigrum L. [3,[6][7][8], not only due to its large abundance in Veratrum nigrum L., but also due to its distinct pharmacological activities including anti-tumor, anti-hypertension, antiplatelet activities [9][10][11], anti-accelerator action on heart, anti-Alzheimer's disease, antimicrobial and anti-inflammatory activities [12][13][14]. Among these reported activities of veratramine, the anti-hypertension effect was the more predominant.…”

Improved Extraction and Complete Mass Spectral Characterization of Steroidal Alkaloids from Veratrum Californicum

“…The principle ion found in peak 3 (410.31 m/z, [M+H] + ) featured the same dominant M-114 ion upon fragmentation as the veratramine standard. Similarly, the M-114 (457.3 m/z) fragment dominates the MS/MS spectrum of the principle ion present in peak 1 (572.0 m/z) and is consistent with the loss of the E-ring in veratrosine.Cyclopamine and cycloposine do not have an aromatic D-ring, but do feature a comparably fragile ether bridge between carbons 17 and 23, which leads to greater fragmentation of the steroidal scaffold than is observed for steroidal alkaloids that do not have the ether bridge[25]. Two dominant ions obtained upon fragmentation of the cyclopamine standard were M-252 and M-250 (157.1 and 159.1 m/z respectively), which are likely the result of loss of water from carbon 3 in conjunction with cleavage through the C-ring.…”