Efficient purification of low molecular weight nitrogen polar compounds from the skin of Bufo bufo gargarizans Cantor by reversed-phase high performance liquid chromatography with a polar-copolymerized C18 stationary phase

Abstract

Cinobufacini (Huachansu) injections have been widely used for the treatment of various cancers in clinical applications in China. As an aqueous extract of the skin of Bufo bufo gargarizans Cantor (toad skin), cinobufacini contains many polar compounds; however the chemical profile of these polar compounds is still unclear, which increases the risk of pharmacological safety. There are very few studies on the separation and purification of polar components from toad skin, especially in the preparative scale. In this study, a unique method based on a homemade polar-modified C18 stationary phase was developed and successfully applied to separate polar compounds from toad skin. Compared with previous purification systems based on the C18 stationary phase, the poor retention problem of polar compounds was well resolved through the use of the polar-modified C18 column and good resolutions were also achieved. Seven compounds were purified and five of them (uracil, hypoxanthine, 3-hydroxy-4H-pyrazolo[4,5-d]pyridazin-7(1H)-one, thymine and bufothionine) were identified by MS, ¹H NMR and ¹³C NMR. In addition, 3-hydroxy-4H-pyrazolo[4,5-d]pyridazin-7(1H)-one was identified as a new compound and hypoxanthine was found from the skin of Bufo bufo gargarizans Cantor for the first time. These results indicate that this method shows promise for the separation of polar compounds in toad skin.