Chemical profiling and cytotoxicity assay of bufadienolides in toad venom and toad skin
Qiong Meng1, Lee-Fong Yau1, Jing-Guang Lu1
1State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macau, China.
Ethnopharmacological Relevance:
Toad venom and toad skin have been widely used for treating various cancers in China. Bufadienolides are regarded as the main anticancer components of toad venom, but the difference on composition and anticancer activities of bufadienolides between toad venom and toad skin remains unclear.
Methods:
Fractions enriched with free and conjugated bufadienolides were prepared from toad venom and toad skin. Bufadienolides in each fraction were comprehensively profiled by using a versatile UHPLC-TOF-MS method. Relative contents of major bufadienolides were determined by using three bufogenins and one bufotoxin as marker compounds with validated UHPLC-TOF-MS method. Furthermore, cytotoxicity of the fractions was examined by MTT assay.
Results:
Two fractions, i.e., bufogenin and bufotoxin fractions (TV-F and TV-C) were isolated from toad venom, and one bufotoxin fraction (TS-C) was isolated from toad skin. Totally 56 bufadienolides in these three fractions were identified, and 29 were quantified or semi-quantified. Bufotoxins were identified in both toad venom and toad skin, whereas bufogenins exist only in toad venom. Bufalin-3-conjugated bufotoxins are major components in toad venom, whereas cinobufotalin and cinobufagin-3-conjugated bufotoxins are main bufotoxins in toad skin. MTT assay revealed potent cytotoxicity of all the fractions in an order of TV-F>TV-C>TS-C.
Conclusions:
Our study represents the most comprehensive investigation on the chemical profiles of toad venom and toad skin from both qualitative and quantitative aspects. Eight bufotoxins were identified in toad skin responsible for the cytotoxicity for the first time. Our research provides valuable chemical evidence for the appropriate processing method, quality control and rational exploration of toad skin and toad venom for the development of anticancer medicines.
Insights
Toad venom and skin contain bufadienolides with anticancer properties. This study identified bufotoxins in toad skin, revealing their cytotoxic potential for developing new cancer medicines.
Area of Science:
- Traditional Chinese Medicine
- Pharmacology
- Analytical Chemistry
Background:
- Toad venom and skin are traditional remedies for cancer in China.
- Bufadienolides are the primary anticancer compounds, but their composition and activity differences between venom and skin are unclear.
Purpose of the Study:
- To comprehensively compare the bufadienolide composition and anticancer activities of toad venom and toad skin.
- To identify key compounds in toad skin responsible for its therapeutic effects.
Main Methods:
- Preparation of bufadienolide-enriched fractions from toad venom and skin.
- Profiling and quantification of bufadienolides using UHPLC-TOF-MS.
- Assessment of fraction cytotoxicity via MTT assay.
Main Results:
- Identified 56 bufadienolides, quantifying 29. Bufotoxins found in both venom and skin; bufogenins only in venom.
- Bufalin-3-conjugated bufotoxins dominate toad venom; cinobufotalin and cinobufagin-3-conjugated bufotoxins are key in toad skin.
- All tested fractions exhibited potent cytotoxicity, with toad venom fractions showing higher activity.
Conclusions:
- This study provides the most comprehensive chemical profile of toad venom and skin bufadienolides to date.
- Eight bufotoxins in toad skin were identified as cytotoxic for the first time.
- Results offer crucial chemical insights for processing, quality control, and developing anticancer drugs from toad skin and venom.
