Abstract
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.
Highlights
The active ingredients in herbal extracts are separated into individual components, by fractionation, desalting, and followed by high-performance liquid chromatography (HPLC)
prostaglandin E1 (PGE1) and PGE2 were purchased from Cayman chemicals (Ann Arbor, MI), and the Fluo8-AM was from ABD-Bioquest (Sunnyvale, CA)
Establishing the stable cell line expressing the EP1 receptor The four subtype receptors which mediate the PGE2pathological functions that cause pain, inflammation, and cancer are the most attractive molecules that can be used as targets for developing the generation of NSAIDs
Summary
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. Various studies have shown the involvement of PGE2 via its EP receptors in growth, differentiation and metastasis of cancer; there are no therapeutic ligands available for these receptors [4,5,6,7]. PGE1 has been used therapeutically in peripheral vascular diseases [8,9,10], and its importance as a potential ligand in cancer cannot be overlooked. We chose to test our herbal extracts on the EP1 subtype receptor since it couples to calcium, which can be used for detecting the stimulation and inhibition of the receptor
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