Research On Isolation And Purification Of Acety-lcholinesterase Inhibitors From Tradional Spices

Chinese traditional spices are widely used in foods all over the world.In addition to flavoring functions,they also have other biological activities,such as scavenging oxygen free radicals,antibacterial,anti-inflammatory,anti-tumor,hypolipidemic,prevention and treatment of atherosclerosis,protection of the nervous system,anti-aging dementia and other physiological functions.In this study,the acetylcholinesterase inhibitory activity of traditional spices was studied.At the same time,the extraction,separation and purification of Acetylcholinesterase inhibitory active ingredients in spice plants were systematically studied.On the other hand,a spectral analysis method was established to determine the chemical constituents of the isolated samples from traditional spices.The main findings and results are as follows:?1?Seven different traditional spices were selected for further study,including Clove,Fennel,Anise,Pepper,Prickly ash,Mustard and Cinnamon.Compared seven samples traditional spices extract under three different extraction methods,by measuring acetylcholinesterase inhibition we found that the solvent reflux extraction was generally high with acetylcholinesterase inhibition.With the same extraction conditions,it was found that the Acetylcholinesterase inhibition activity of Zanthoxylum bungeanum was higher in seven spices,and the Acetylcholinesterase inhibition rate was 45.47%,which was optimized for further study.?2?Based on single factor experiment and orthogonal optimization experiment,the effects of three main factors of the inhibitors extract of Zanthoxylum bungeanum Maxim were studied.The optimum conditions of the extraction process are as follows: ethanol concentration 80%,solid-liquid ratio 1:12,extract 60 min at room temperature,and extract for 120 min at 70?.Under the optimum conditions,determine the Acetylcholinesterase inhibition rate of the extract of Zanthoxylum bungeanum Maxim.was 67.42%.?3?Four different polarity solvents of petroleum ether,methylene chloride,ethyl acetate,and n-butanol were used to franctional extraction of Ethanol extract.The result showed that the ethyl acetate extracted components had the highest inhibitory activity in acetylcholinesterase.By using AB-8 macroporous resin adsorption to preliminarily separate acetylcholinesterase inhibitors in the ethyl acetate fraction,we determined Fr.1,Fr.2,and Fr.3 three components were with higher inhibitory activity.By using Silica gel column chromatography to further purification,we found that Fr.1 had highest inhibitory activity and obtained two highly active components?component A and component B?with further separation.By using Sephadex-G15 column chromatography to further purification,we found that component A showed lower inhibition rate and was lower than 30%,so we gave up further collection,While from component B we obtained two highly active components?component B1 and component B2?with further separation.Component B2 was mainly collected for subsequent purification and structural identification.By using the thin-layer chromatography for preliminary identification of B2,and it was determined that the obtained component was mainly flavonoids.By using TLC selected elution solvents and repeated elution on the column.Finally,two monomer compounds were separated with high activity and high purity.?4?By using UV,IR,¹H-NMR and ¹³C-NMR,two monomers were elucidated as catechins and quercetin-3-galactopyranoside.Compared with the standard samples by thin layer chromatography confirm the conjecture was correct.