An agonist sensitive, quick and simple cell-based signaling assay for determination of ligands mimicking prostaglandin E2 or E1 activity through subtype EP1 receptor: Suitable for high throughput screening

BMC Complementary and Alternative Medicine

Research Article

Ke-He Ruan¹  Annirudha J Chillar¹  Parastoo Karimi¹  Kathy Tang² 

[1] Centre for Experimental Therapeutics and Pharmacoinformatics, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, 77004, Houston, Texas, USA;TR Acupuncture and Herbal Clinic, 77005, Houston, TX, USA;

关键词: HEK293 Cell;    Calcium Signal;    High Throughput Screening;    Stable Cell Line;    Herbal Extract;   

DOI  :  10.1186/1472-6882-11-11

 received in 2010-10-13, accepted in 2011-02-07,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundConventionally 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 prostaglandin E1 (PGE1) and E2 (PGE2) can be identified in the water-soluble non-purified fraction. PGE1 is a potent anti-inflammatory molecule used for treating peripheral vascular diseases while PGE2 is an inflammatory molecule.MethodsWe used cell-based assays (CytoFluor multi-well plate reader and fluorescence microscopy) in which a calcium signal was generated by the recombinant EP1 receptor stably expressed in HEK293 cells (human embryonic kidney). PGE1 and PGE2 were tested for their ability to generate a calcium signal. Ninety-six water soluble fractions of Treasures of the east (single Chinese herb dietary supplements) were screened.ResultsAfter screening, the top ten stimulators were identified. The identified herbs were then desalted and the calcium fluorescent signal reconfirmed using fluorescence microscopy. Among these top ten agonists identified, seven stimulated the calcium signaling (1-40 μM concentration) using fluorescence microscopy.ConclusionsFluorescence 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, PGE1 and PGE2.

【 授权许可】

Unknown   
© Chillar et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.