Screening And Isolation Of Alcohol Dehydrogenase Inhibitors From Chinese Herbal Medicine By Activity-guided Fractionation

The liver enzyme alcohol dehydrogenase (ADH) is responsible for the oxidative metabolism of ethanol. Its inhibitors could suppress the acetaldehyde accumulation in alcohol-hypersensitive alcoholics by inhibiting the activity of ADH. ADH inhibitors also play an important role in the treatment of human methanol and ethylene glycol poisoning. ADH inhibitors have been developed that have much side effects and too expensive. Recently, active compounds were found in natural plants, which showed fewer side effects, higher security and more suitable for the requirements of the patients. As we know, the chemical composition of natural medicinal plants is very complex including hundreds and even thousands of different compounds. However, only a few such compounds are responsible for pharmaceutical and/or toxic effects. The conventional activity-guided fractionation of complex plant extracts is a time-consuming, labor intensive and expensive process. Screening and isolation of active compounds from complex mixture is one of the hotspots and difficult problems of rapid screening research.In recent new methods of screening active compounds from complex mixtures, activity-guided fractionation has attracted more and more interest of the researchers. Therefore, the aim of this dissertation is to screen and isolate of ADH inhibitors from Chinese herbal medicine by activity-guided fractionation.Our chief research work is as follows:1. Fe3O4@SiO2nanoparticles was prepared, and used as carrier, with the aid of glutaraldehyde as cross-linking agent to immobilize ADH. The amount of bound ADH onto magnetic nanoparticles was measured. The temperature and pH values were optimized to determine their influences on the activity of immobilized ADH. It was demonstrated that compared with solution enzyme, the pH range of enzyme activity and the thermo stability of immobilized enzyme were both increased, and of which the repeating utilization ratio and the storage stability were satisfactory. The Fe3O4@SiO2-ADH functionalized magnetic nanoparticles was used to screen ADH inhibitors from Glycyrrhiza Uralensis root extract. The reaction conditions were optimized. Nine ADH inhibitors were revealed and identified, among which seven were first screened as ADH inhibitors.2. A valid method based on ultrafiltration combined with high-speed countercurrent chromatography was used to rapid screen and isolate ADH inhibitors from Glycyrrhiza Uralensis root extract for the first time. Experiments were carried out to optimize binding conditions including ADH concentration, incubation time, temperature, and pH. By comparing the chromatograms, three compounds were found possessing ADH binding activity in Glycyrrhiza uralensis root. Under the target-guidance of ultrafiltration combined with high performance liquid chromatography experiment, liquiritin, isoliquiritin and liquiritigenin were separated by high-speed countercurrent chromatography using ethyl acetate-methanol-water (5:1:4) as the solvent system. Isoliquiritin showed strongest inhibitory activity with IC5o of8.95μM.3. A new combinative method based on ultrafiltration combined with high performance liquid chromatography was developed to screen ADH inhibitors from Desmodium styracifolium extract. Experiments were carried out to optimize binding conditions including ADH concentration, incubation time, temperature, and pH. Compared with standard sample, two ADH inhibitors (aromadendrin and formononetin) were revealed and identified. Aromadendrin was first isolated as ADH inhibitor.