In this study, an approach based on triple-color fluorescence probes was developed for screening potential nephroprotective bioactive substances. Three fluorescent probes (i.e. FDA, MTR and Hoechst 33342) were used to label HK-2 cells injured by doxorubicin hydrochloride, and cellular fluorescence images were subsequently acquired and analyzed by a cellular-fluorescence image microscopy platform. The established method was applied to screening 53components of Carthami Flos, and three components C17, C18and C19 were found to exhibit nephroprotective effects against doxorubicin hydrochloride induced injury on HK-2 cells. Eight compounds (i.e. hydroxysafflor yellow A, 6-hydroxykaempferol-3-O-rutinoside-6-O-glucoside, 6-hydroxykaempferol-3, 6-di-O-glucoside or 6-hydroxykaempferol-6, 7-di-O-glucoside, 6-hydroxykaempferol-3-O-rutinoside, 6-hydroxykaempferol-3-O-glucoside or 6-hydroxykaempferol-7-O-glucoside, rutin, isoquercetin, and kaempferol-3-O-rutinoside) in components C17, C18 and C19 were preliminarily identified by liquid chromatography-mass spectrometry (LC-MS). Isoquercetin, rutin, kaempferol-3-O-rutinoside, and hydroxysafflor yellow A were confirmed by comparing with reference substances, Further study indicated that these four compounds had moderate nephroprotective effects, while isoquercetin showed a significant nephroprotective effect in a dose-dependent manner. These results suggest that isoquercetin, rutin, kaempferol-3-O-rutinoside and hydroxysafflor yellow A might be the nephroprotective bioactive substances in Carthami Flos.
- Carthami Flos
- Fluorescence probes
- Screening bioactive substances