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Toad venom (Chansu), a traditional Chinese medicine (TCM), has been widely used for treating various cancer. However, it is considerably difficult to evaluate the quality of Chansu due to its complex chemical compositions. Hence, finding the characteristic ingredients and developing a scientific and comprehensive quality evaluation method are essential for guaranteeing the safety and efficacy of Chansu. In this paper, the chemical composition database of Chansu was successfully established and HPLC-ESI-Q-TOF-MS/MS was applied for chemical profiling of the ingredients in Chansu. In total, 157 compounds were identified, including 22 amino acids, 8 alkaloids, 54 bufogenins, 63 bufotoxins, and 10 other compounds. Furthermore, HPLC fingerprints and quantitative analysis of its multicomponent were successfully developed to evaluate the quality consistency of Chansu from different origins. The results suggested that the HPLC fingerprint of Chansu could be divided into an amino acid and alkaloid region, as well as a bufogenins and bufotoxins region. The fingerprint profile of Chansu from different geographical origins were different, indicating that its quality was affected by the geographical factors. In addition, seven characteristic peaks were selected as the quantitative markers to evaluate the quality of the Chansu. The Kruskal–Wallis test illustrated that the contents of seven bufogenins in Chansu were significantly (p < 0.01) different among different origins. The total contents of the seven compounds ranged from 100.40 to 169.22 mg/g in 20 batches of Chansu samples. This study demonstrated that integrating HPLC-ESI-Q-TOF-MS/MS, HPLC fingerprints, and multicomponent quantitative analysis coupled with chemometrics was a comprehensive and reliable strategy for evaluation of Chansu in both qualitative and quantitative aspects. In addition, our study represented the most comprehensive characterization on the chemical compositions of Chansu, which could provide important reference information for the discovery of potential bioactive compounds.

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(1) Background: Toad venom (Bufonis Venenum, known as ‘Chansu’ in Chinese), the secretion of the ear-side gland and skin gland of Bufo gargarizans cantor or Duttaphrynus melanostictus Schneider, has been utilized to treat several diseases in China for thousands of years. However, due to the chemical variability of the components, systematic chemical composition and the key pharmacophores in toad venom have not yet fully understood. Besides, it contains a variety of effective compounds with different physiological activity and chemotypes, mainly including alkaloids, bufogenins, bufotoxins, and so on. The recent pharmacological researches have demonstrated that several bufogenins have remarkable pharmacological effects, such as anti-inflammatory, analgesic effects, and anti-tumor effects. Aim of the study: To identify the bioactive compounds and pharmacophores originating from toad venom based on analyzing spectrum-effect relationship by chemometrics and to explore the anti-cancer mechanism primarily. (2) Materials and methods: Fingerprint of the 21 batches of samples was established using HPLC (High Performance Liquid Chromatography). The anti-tumor activity of extracts were determined by in-vitro assays. Chemometric analysis was used to establish the spectrum-effect model and screen for active ingredients. Pharmacodynamic tests for the screened active compound monomers were conducted with in-vitro assays. Further anti-tumor mechanisms were investigated using western blot and flow cytometry. (3) Results: The established spectrum-effect model has satisfactory fitting effect and predicting accuracy. The inhibitory effect of major screened compounds on lung carcinoma cells A549 were validated in vitro, demonstrating that arenobufagin, telocinobufogenin, and cinobufotalin had significant anti-tumor effects. Through further investigation of the mechanism by western blotting and flow cytometry, we elucidated that arenobufagin induces apoptosis in A549 cells with the enhanced expression of cleaved PARP (poly (ADP-ribose) polymerase). These results may provide valuable information for further structural modification of bufadienolides to treat lung cancer and a method for discovery of anti-tumor active compounds. Conclusions: Our research offers a more scientific method for screening the principal ingredients dominating the pharmacodynamic function. These screened compounds (arenobufagin, etc.) were proven to induce apoptosis by overactivation of the PARP-pathway, which may be utilized to make BRCA (breast cancer susceptibility gene) mutant cancer cells more vulnerable to DNA damaging agents and kill them.