An agonist sensitive, quick and simple cell-based signaling assay for determination of ligands mimicking XXXX or E1 activity through subtype EP1 receptor: Suitable for high throughput screening.

@entity1095 @entity1 @entity1421 @entity5015 @entity460
BACKGROUND: Conventionally the active ingredients in herbal extracts are separated into individual components, by fractionation, desalting, and followed by high-performance liquid chromatography (HPLC). In this study we have tried to directly screen water-soluble fractions of herbs with potential active ingredients before purification or extraction. We propose that the herbal extracts mimicking @entity5015 ( @entity5015 ) and E(2) ( @entity1421 ) can be identified in the water-soluble non-purified fraction. @entity5015 is a potent anti-inflammatory molecule used for treating @entity1095 while @entity1421 is an inflammatory molecule. METHODS: We used cell-based assays (CytoFluor multi-well plate reader and fluorescence microscopy) in which a @entity460 signal was generated by the recombinant EP(1) receptor stably expressed in HEK293 cells ( @entity1 embryonic kidney). @entity5015 and @entity1421 were tested for their ability to generate a @entity460 signal. Ninety-six water soluble fractions of Treasures of the east (single Chinese herb dietary supplements) were screened. RESULTS: After screening, the top ten stimulators were identified. The identified herbs were then desalted and the @entity460 fluorescent signal reconfirmed using fluorescence microscopy. Among these top ten agonists identified, seven stimulated the @entity460 signaling (1-40  M concentration) using fluorescence microscopy. CONCLUSIONS: Fluorescence microscopy and multi-well plate readers can be used as a target specific method for screening water soluble fractions with active ingredients at a very early stage, before purification. Our future work consists of purifying and separating the active ingredients and repeating fluorescence microscopy. Under ordinary circumstances we would have to purify the compounds first and then test all the extracts from 96 herbs. Conventionally, for screening natural product libraries, the procedure followed is the automated separation of all constituents into individual components using fractionation and high performance liquid chromatography. We, however, demonstrated that the active ingredients of the herbal extracts can be tested before purification using an agonist sensitive, quick and simple cell-based signaling assay for ligands mimicking the agonists, @entity5015 and @entity1421 .
