| Screening bioactive compounds from traditional Chinese medicine is one of the most useful methods to discover new drug. Classic screening methods such as bioassay-guided screening are labor intensive, time consuming and low efficient. Moreover, the long process of isolation and purification may lead to the loss of activity due to dilution or decomposition and separated compounds that are inactive. In recent years, chemical biology based screening techniques that directly identifying potential ligands by specific ligand-receptor binding from unpurified natural products extracts have been developed, such as ultrafiltration, magnetic beads, turbulent chromatography, and bioaffinity cell membrane chromatography.However, the methodology and applied domain of those approaches were rarely studied.This thesis established five chemical biology based approaches including a magnetic bead based method for screening a-glucosidase inhibitors, two ultrafiltration based methods for screening a-amylase and15-lipoxygenase inhibitors, a hollow fiber based method for screening lipase inhibitors and a multi-target based method for screening maltase, invertase and lipase inhibitors. More than ten bioactive compounds were identified from mulberry leaves, lotus leaf, hawthorn leaf, Senecio cannabifolius and San-Ye-Tang-Zhi-Qing.The main contents include:1. Characterization the constituents of San-Ye-Tang-Zhi-Qing using high performance-liquid chromatography coupled with high resolution mass spectrometry.25compounds in formulae San-Ye-Tang-Zhi-Qing were detected, including six flavonoids, eight flavonol glycosides, nine phenolic acids. 2. Chemical investigation of the water extracts from the Senecio cannabifolius Less, led us to find fourteen known compounds and two new ones. Their structures were determined as (E,4R)-4-hydroxy-4,5,5-trimethyl-3-(3-oxobut-1-enyl)cyclohex-2-enone(1) and (E)-4-((1S,3R,4R)-1-hydroxy-4,5,5-trimethyl-7-oxabicyclo[4.1.0] heptan-1-yl)but-1-en-3-one(2), respectively.3. An alpha-glucosidase coated magnetic beads based screening approach was established and had been applied to the extract of Morus Alba. The extract of Morus alba, a Chinese medicinal plant, was screened for potential ligands of alpha-glucosidase using the established method. The structures of potential active compounds were identified via liquid chromatography-mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR). The results demonstrated that two flavonoids (isoquercitrin and astragalin) could bind alpha-glucosidase, which was confirmed via conventional alpha-glucosidase inhibitory assay.4. An ultrafiltration liquid chromatography-mass spectrometry based screening approach was developed and had been applied to screening a-amylase inhibitors from the extract of hawthorn leaf. Three active compounds were screened out as a-amylase inhibitors from the extract of hawthorn leaf, a-amylase inhibitory assay validated the activity of the three compounds. Meanwhile, the ultrafiltration based approach using15-lipoxygenase as a target was applied to screening15-lipoxygenase inhibitors from Senecio cannabifolius and an active compound was found.15-lipoxygenase inhibitory assay verified the activity of the compound.5. A lipase adsorbed hollow fiber based screening approach was established and had been applied to screen ligands from the extract of lotus leaf. The hollow fiber based approach was applied to screen lipase inhibitors from extract of lotus leaf and three active compounds were found. Lipase inhibitory assay validated the activity of the three compounds.6. A sequential magnetic bead-based multi-target screening approach was developed and had been applied to the’San-Ye-Tang-Zhi-Qing’formulae. Maltase, sucrase and lipase were selected as screening targets to establish a multi-target screening approach. The multi-target screening approach was applied to screening bioactive compounds from the’San-Ye-Tang-Zhi-Qing’formulae. Conventional inhibitory assay verified the activity of five compounds including2,3,4,6-tetra-O-galloyl-D-glucopyranose,1,2,3,4-tetra-O-galloyl-D-glucopyranose,1,2,3,4,6-penta-O-galloyl-D-glucopyranose, quercetin-3-O-β-D-glucuronide and isoquercitrin.In summary, this thesis established a class of chemical biology-based screening approaches, including ultrafiltration, magnetic beads, hollow fibers and multi-target based screening approaches, for identifying bioactive compounds from traditional Chinese medicine. These screening approaches had the following advantages:The screening efficiency was high and the cost was low. The screening approach doesn’t require cumbersome and complex separation and preparation process. By intergrating the screening approach with mass spectrometry and nuclear magnetic resonance, the structures of bioactive compounds could be elucidated. |
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