State 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.
Beijing Increase Pharm. Co. Ltd., Beijing, China.
J Ethnopharmacol. 2016 Jul 1;187:74-82. doi: 10.1016/j.jep.2016.03.062. Epub 2016 Apr 6.
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.
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.
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.
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.
在中国，蟾蜍毒液和蟾皮被广泛用于治疗各种癌症。蟾毒内酯被认为是蟾蜍毒液的主要抗癌成分，但蟾蜍毒液和蟾皮中蟾毒内酯的组成和抗癌活性仍不清楚。
从蟾蜍毒液和蟾皮中制备富含游离和结合型蟾毒内酯的馏分。采用通用的 UHPLC-TOF-MS 方法对各馏分中的蟾毒内酯进行综合分析。采用三种蟾毒配基和一种蟾毒素作为标记化合物，通过经验证的 UHPLC-TOF-MS 方法，确定主要蟾毒内酯的相对含量。此外，通过 MTT 法检测馏分的细胞毒性。
从蟾蜍毒液中分离出两个馏分，即蟾毒配基和蟾毒素馏分（TV-F 和 TV-C），从蟾皮中分离出一个蟾毒素馏分（TS-C）。这三个馏分中共鉴定出 56 种蟾毒内酯，其中 29 种进行了定量或半定量分析。蟾毒素在蟾蜍毒液和蟾皮中均有发现，而蟾毒配基仅存在于蟾蜍毒液中。蟾蜍毒液中主要的结合型蟾毒素是蟾毒配基-3-结合型蟾毒素，而蟾皮中主要的蟾毒素是华蟾毒精和华蟾毒它灵-3-结合型蟾毒素。MTT 法显示所有馏分均具有较强的细胞毒性，其顺序为 TV-F>TV-C>TS-C。
本研究从定性和定量两个方面代表了对蟾蜍毒液和蟾皮化学成分的最全面研究。首次在蟾皮中鉴定出 8 种蟾毒素，这些毒素与细胞毒性有关。本研究为蟾蜍毒液和蟾皮的合理加工方法、质量控制和合理开发抗癌药物提供了有价值的化学依据。
