Identification of anti-tumor constituents from toad skin and toad venom by UPLC-QTOF/MS in-depth chemical profiling combined with bioactivity-based molecular networking

Toad skin (TS) and toad venom (TV) are two different traditional Chinese medicine (TCM) prepared from Bufo bufo gargarizans Cantor and Bufo melanostictus Schneider. Both of them were used in the treatment of ulcers, and now they are regarded as animal drugs with anti-tumor activity. However, the comparison analysis between their compositions and anti-tumor active ingredients has not been elucidated clearly. In this study, technology of ultra-performance liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) and bioactivity-based molecular networking were applied to investigate the anti-tumor active ingredients of TS and TV. A method of collision energies-relied mass spectrometry combined with molecular networking was initially developed for identifying their chemical components. A combination of Cytoscape and molecular networking was then employed to analyze the prototype constitutes of TS absorbed in rat serum. In addition, cytotoxicity test was applied to evaluate their anti-proliferative activities against human gastric cancer (GC) cells. As a result, a total of 162 compounds were tentatively characterized, and four structural types existed in both TS and TV, including bufogenins, bufotoxins, indolealkylamines (IAAs), and acyl-arginine. Cyclic dipeptides could only be detected in TS, and the total number of bufogenins and bufotoxins in TV was higher than that of TS. Both of them significantly inhibited the viability of SGC7901 cells, and 70% methanol eluate of TS was the most potential active fraction. 44 prototype compounds in TS were identified, from which three types of potential tumor cytotoxic components, including bufogenins, IAAs, and cyclic dipeptides, were predicted. In human GC cells SGC7901, a mixture of nine bufogenins, including bufotalin, cinobufotalin, gamabufotalin, telocinobufagin, cinobufagin, bufarenogin, arenobufagin, hellebrigenin, and desacetylcinobufotalin, was conducted as bioactive components, which exerted a significant inhibitory effect on the cell viability of SGC7901 cells with a half maximal inhibitory concentration (IC50) value of 0.23 (0.19–0.27, 95%CI) μg/mL. Comparison of their chemical composition and anti-tumor activity reveals that TS might be able to partially replace TV, whilst bufogenins are the potential active ingredients