The Rapid Isolation,Purification Of The Effective Components In Chinese Herbal And The Study Of The Fingerprinting

The search for lead compounds from Chinese herbal medicine is one of the most important approaches in international drug discovery activities.On one hand,it needs extraction,separation and purification of active ingredients from Chinese herbal medicine to establish the material foundation for pharmacological research and innovative medicines,on the other hand,the quality control research is also needed to protect the efficacy of Chinese herbal medicines.Therefore,an urgent requirement for the methods of extraction,separation,purification and quality control of Chinese herbal medicine ingredient is coming to being.Taking Uncaria rhynchophylla(Miq.)Jacks,Lonicera japonica Thunb and Lonicera macranthoides Hand-Mazz as research subject,this paper discussed extraction,separation,and purification methods of active ingredients,the main component measurements and elemental fingerprint,and made some of the innovative achievements.These results have important implications for identification,provenance and product quality testing for Chinese herbal medicines.The main results are summarized as follows:(1)The contents of Uncaria rhynchophylline,isorhynchophylline in Uncaria rhynchophylla harvested at different time during the growing period were determined by HPLC.The results show that the best harvest time of Jianhe Uncaria rhynchophylla is in late November.The determine method of chlorogenic acid,luteoloside and secoxyloganin in Lonicera japonica Thunb were studied.The optimum harvest period is dabai period or erbai period.The contents of macranthoides saponin B and asperosaponin B in Lonicera macranthoides Hand-Mazz at different patterns were determined,and the results show that contents under technology integration planting pattern are higher than conventional planting pattern.(2)The effects of extraction rate with different solvents,methods and time through different extraction methods on major component in Lonicera japonica Thunb were compared.The results show that the extraction rate with heating reflux extraction is higher than ultrasound and cold soak by using 70% ethanol or water.The best traditional extraction process is heating reflux for 0.5h,2 times with pure water,which can extract more than 95% of the active ingredients.A new UHPE-HPLC method was established to determine 10 active ingredients in Lonicera japonica Thunb.The optimum extraction conditions were that under these conditions of 60% methanol,400 MPa pressure,3 minutes and with a solid-liquid ratio 1:30(g/m L),the extraction rate can reach 57.62 mg/g.Meanwhile,the linear,recovery rates,LODs and LOQs of this method meeting the requirement of experiment.The microstructure characterization of the extracted samples indicate Ultrahigh-pressure Extraction method is more rapid and efficient.(3)A UPLC-TOF-MS method was established to quickly analyze and identify compounds in Lonicera macranthoides Hand-Mazz.With this method,40 compounds can be identified from Lonicera macranthoides Hand-Maz,which can be used as precise identification technology.(4)A procedure was developed to separate chlorogenic acid,luteoloside,secoxyloganin and loganin from Lonicera japonica Thunb,by which the 4 compounds can be obtained with high purity.And,a method of HSCCC was established to separate and prepare chlorogenic acid.With the solvent system composed of ert-butyl methyl ether: n-butanol: acetonitrile: water(2:2:1:5,v/v),5% diisooctyl phosphate ester being added in stationary phase and 20 mmo L/L hydrochloric acid being added in mobile phase,the purity of chlorogenic acid acquired is 99.70%.Another new HSCCC method was established to separate and prepare luteoloside,by which the purity of luteoloside acquired is 98.87% with the solvent system composed of ethyl acetate: ethanol: acetic acid: water(4:1:0.25:5,v/v).In this study,HSCCC was first applied to the separating and purifying luteoloside from Lonicera japonica Thunb,which is expected to solve the problem of lacking reference in quality control of Lonicera japonica Thunb.(5)The p H-Zone refining counter-current chromatography was firstly successfully applied to the preparative isolation and purification of 6 alkaloids in the ethanol extracts of Uncaria macrophylla Wall.Because of the low content of alkaloids(about 0.2%,w/w)in U.macrophylla Wall,the target compounds were enriched by p H-zone refining counter-current chromatography using a two-phase solvent system composed of petroleum ether: ethyl acetate: isopropanol: water(2:6:3:9,v/v),adding 10 mmo L/L triethylamine in organic stationary phase and 5 mmo L/L hydrochloric acid in aqueous mobile phase.Then p H-zone refining counter-current chromatography using the other two-phase solvent system was used for final purification.6 target compounds were finally isolated and purified by following two-phase solvent system composed of methyl tert-butyl ether(MTBE)-acetonitrile-water(4:0.5:5,v/v),adding triethylamine(TEA)(10 mmo L/L)in the organic phase and HCl(5 mmo L/L)in aqueous mobile phase.The separation of enriched total alkaloids yields 6 alkaloids with purities above 96%.Compared to conventional production,this method has proven to be fast and efficient,for it solves the technical difficulties on alkaloids enrichment and by which more alkaloids can be obtained.Also it can be regarded as a new technology to separate and purify the active ingredients in Uncaria alkaloids,and has important significance for effective substances and quality control of Chinese herbal medicine as well.Also,a new method was established to separate and purify alkaloid in Picrasma quassioides(D.Don)Benn by using p H-Zone refining counter-current chromatography.Compared with conventional method,this new method has advantages of large injection amount and higher alkaloid purity.(6)A simple and reliable ICP-MS method was used to establish elements fingerprints of Uncaria rhynchophylla from Jianhe,Tianzhu and Jiangyou respectively,also elements fingerprints of Lonicera macranthoides Hand-Mazz from Suiyang,Danzhai and Taian were established.The result through Cluster analysis and PCA analysis shows that samples from different habitats can be classified into a different category.The method established in this paper is of great importance in variety identification,origin traceability and quality control of Chinese herbal medicines.