Background and purpose: We report the development of a very efficient cell-based high throughput screening (HTS) method, which utilizes a novel bio-sensor that selectively detects apoptosis based on the fluorescence resonance energy transfer (FRET) technique. Experimental approach: We generated a stable HeLa cell line expressing a FRET-based bio-sensor protein. When cells undergo apoptosis, they activate a protease called 'caspase-3'. Activation of this enzyme will cleave our sensor protein and cause its fluorescence emission to shift from a wavelength of 535 nm (green) to 486 nm (blue). A decrease in the green/blue emission ratio thus gives a direct indication of apoptosis. The sensor cells are grown in 96-well plates. After addition of different chemical compounds to each well, a fluorescence profile can be measured at various time-points using a fluorescent plate reader. Compounds that can trigger apoptosis are potential candidates as anti-cancer drugs. Key results: This novel cell-based HTS method is highly effective in identifying anti-cancer compounds. It was very sensitive in detecting apoptosis induced by various known anti-cancer drugs. Further, this system detects apoptosis, but not necrosis, and is thus more useful than the conventional cell viability assays, such as those using MTT. Finally, we used this system to screen compounds, isolated from two plants used in Chinese medicine, and identified several effective compounds for inducing apoptosis. Conclusions and Implications: This FRET-based HTS method is a powerful tool for identifying anti-cancer compounds and can serve as a highly efficient platform for drug discovery.

A high-performance liquid chromatographic (HPLC) method coupled with ultraviolet (UV) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF/MS) was established for simultaneous qualitative and quantitative determination of nine phenolic acids and six diterpenoids in Radix et Rhizoma Salviae Miltiorrhizae (RRSM). The optimal chromatographic conditions were achieved on a Zorbax C 18 column by gradient elution with 0.1% (v/v) aqueous formic acid and acetonitrile as mobile phase at the flow rate of 1.0 mL/min. The detection wavelength at 281 nm was chosen to determine the 15 bioactive components, namely danshensu (1), protocatechuic acid (2), protocatechuic aldehyde (3), caffeic acid (4), rosmarinic acid (5), lithospermic acid (6), salvianolic acid B (7), salvianolic acid A (8), salvianolic acid C (9); dihydrotanshinone I (10), cryptotanshinone (11), tanshinone I (12), methylene tanshiqunone (13), tanshinone IIA (14) and miltirone (15). Additionally, LC-ESI-TOF/MS was used to make definite identification of the constituents in samples in comparison with those reference compounds. The validation of the method included tests of linearity, sensitivity, repeatability, stability and recovery. The proposed method was successfully applied to quantify the 15 components in 21 samples; significant variations were demonstrated in the contents of the samples from diverse species and origins. The developed method could be used to effectively and comprehensively evaluate the quality of RRSM for its clinical safety and efficacy.

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