Abstract
In order to evaluate the quality of Bufonis Venenum commercial herbs, a three-step qualitative and quantitative research study was performed. Firstly, we tried to identify small molecules and peptides in Bufonis Venenum using pre-fractionation chromatography and high-resolution mass spectrometry. The database search of the small molecules and peptides of Bufonis Venenum revealed that the dried venom consisted of free/conjugated-type bufadienolides and peptides with a mass range of 0.4–2 kDa. Secondly, we used partial least squares (PLS) multivariate statistical analysis to screen bufadienolides markers (VIP > 1.5) responsible for the anti-tumor cell activity of Bufonis Venenum, including 21 identified bufadienolides and 7 unknown compounds. It is noticeable that these bufadienolide markers could not be recognized by traditional HPLC-UV based spectrum-effect relationship analysis (correlation coefficient ranging from −0.24 to 0.40). Finally, we proposed a weight coefficient-based corrected total contents of 9 bufadienolides as a quality evaluation indicator, which had good correlation with inhibitory effects on tumor cells of commercial Bufonis Venenum. The correlation coefficient increased from 0.4 to 0.6. Thus, our pre-fractionation chromatography and mass spectrometry strategy had significant advancement over the traditional spectrum–effect relationship method for chemical marker identification. These results could be crucial and helpful in the development of a quality evaluation method that could reflect the pharmacological activity of Bufonis Venenum.
Highlights
In recent years, Chinese medicine has made significant progress in modernization and globalization, the quality evaluation of TCM is still in a relatively simple stage
All fractions were further analyzed by high-resolution mass spectrometry (HRMS)
We proposed a pipeline to globally identify small molecules and peptides in Bufonis Venenum
Summary
Chinese medicine has made significant progress in modernization and globalization, the quality evaluation of TCM (traditional Chinese medicine) is still in a relatively simple stage. The main technical methods to control the quality are still based on traditional traits, microscopic identification and, physical and chemical testing, supplemented by the corresponding content determination [1,2,3]. The development of multi-dimensional chromatography and LC/MS technology has enabled the qualitative identification and quantitative detection of hundreds of compounds from a Molecules 2019, 24, 1943; doi:10.3390/molecules24101943 www.mdpi.com/journal/molecules. The detection methods mentioned above only use the content of one or several components in the medicine as the indicator of quality evaluation. The composition of traditional Chinese medicines is complex and diverse. This method of chemical evaluation does not have specific properties because the “indicative component” of most Chinese medicines is neither its exclusive component nor its active ingredient, while the inherent “multi-component and multi-target”
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