| This paper includes five parts:The first part: summary of the related literatureDocuments about the present research situation in the chemical constituents and pharmacological activities of Corydalis yanhusuo in the last ten years were summed up and analyzed, including the structure of compounds, pharmacodynamics, pharmacokinetic and toxicity. Special attentions were paid to the effect and mechanism of activities in central nervous system, cardiovascular system and Cerebrovascular system. 93 documents were cited.The second part: initial studies on the preparative technology of the medicinal extract and verification of the activityThe preparative technology of alkaloids extract of Corydalis yanhusuo was established by using total alkaloids contents as the evaluating quota. After investigating the adsorptive properties of several kinds of resins, C macroporous absorption resin was selected owning to its reasonable properties of absorption and isolation of the total alkaloids of Corydalis yanhusuo. The ratio of dynamic loading quantity to resin volume was 1.99g crude drug to 1.00ml wet resin. After eluted with alkalescence water (10 times of resin volume), the resin was eluted by W% ethanol with 10 times of resin volume. The resin could be used after regenerated with 95%ethanol and 1%HCl. The technology was tested and verified through 3 batched of samples. The results showed that the preparative technology of the medicinal fraction was reasonable, steady and repetitive. The total alkaloids contents of the medicinal extract were above 50%. The anti-myocardial ischemia extract was gained though comparing the activities of different parts of extracts. The activity of the alkaloids extract was verified though anti-myocardial ischemia tests.The third part: research on the chemical constituents of the medicinal extractThe chemical constituents of anti-myocardial ischemia medicinal extract of Corydalis yanhusuo were researched for the first time. Several purification and separation methods and many kinds of structure elucidation and identification technologies such as UV, IR, ESI-MS, HRMS, 1HNMR, 13CNMR, HMBC, NOESY were used. 11 compounds were isolated, all of which were identified as alkaloids. Two were tertiary alkaloids, nine were quaternary (eight protoberberine quaternary alkaloids, one aporphine quaternary alkaloid). Among them, a new compound was identified as 5, 6-dihydro-10-hydroxy-2, 3, 9-trimethoxy-13-methyl-dibenzo[a,g] quinolizinium alkaloid, which was named 13-methyl-dehydrocorydalmine, and a new natural compound was identified as 5, 6-dihydro-9-hydroxy-2,3,10-trimethoxy-13-methyl- dibenzo[a,g] quinolizinium alkaloid, which was named 13-methyl-palmatrubine. 3 compounds (noroxyhydrastinine, corunine and dehydrocorybulbine) were isolated from Corydalis yanhusuo for the first time. The others alkaloids isolated from the medicinal extract were known alkaloids, which has been isolated from Corydalis yanhusuo. They are tetrahydrocolumbamine, dehydrocorydaline, columbamine, coptisine, palmatine and berberine.The fourth part: qualitative and quantitive analysis of the medicinal extractTLC method was established for the fast qualitative identification of the medicine extraction of Corydalis yanhusuo.The quantitative method of total alkaloids in the medicine extraction of Corydalis yanhusuo was established. The results showed that these methods were accurate and have nice recovery rates and good repetition, which could be used to control the quality of the medicine extraction.For the first time, the HPLC fingerprints of the medicine extraction were established to control the quality of the medicine extraction. The main parameters of the HPLC fingerprints were determined after detecting and analyzing 9 batches of samples. When the detective wavelength was in 335nm, 12 common peaks were found, and the RSD of their relative retention time were 0.10%～1.48%. The RSDs of the relative area of peaks were 3.57%～26.01%. The total area of the common peaks was % in proportion to that of all peaks with RSD=2.28%.The chemical constituents of which the common peaks stood for respectively were analyzed by adding controls to the samples and HPLC-MS-MS technology. 12 common peaks were analyzed, and the main constituents of 11 peaks were identified. 9 of them were quaternary alkaloids. The total area of these 9 peaks was 90% in proportion to that of all peaks. The results further indicated the main constituents in the medicinal extract were quaternary alkaloids.On the basis of these analysis, the methods of simultaneous determination of eight protoberberine quaternary alkaloids (dehydrocorydaline, columbamine, coptisine, palmatine, 13-methyl-dehydrocorydalmine, 13-methyl-palmatrubine, dehydrocorybulbine, berberine) in the medicinal extract of Corydalis yanhusuo were established which was sensitive, fast and reliable. These work provided a basis for developing a novel drug and establishing its scientific, reasonable and strict quality control system in the future.The fifth part: initial study of the absorbed constituents of the medicinal extractHPLC-MS-MS method was employed in the analysis of alkaloids and metabolic in rat plasma samples after oral administration of MEC. The structure of 11 components from MEC were identified or tentatively elucidated. The results showed that most of quaternary protoberbererine alkaloids of MEC can be detected, such as dehydrocorydaline, Palmatine, berberine, etc. This mean they can be absorbed by oral administration. The peaks of metabolic HPLC fingerprint can be used as the active constituents and marked components for the quality control of the medicinal extract of Corydalis yanhusuo. Furthermore, the glucuronide conjugates of alkaloids were observed in the plasma.According to the results of qualitative analysis, variations of the concentration of 4 identified components in vivo and the distribution in issues were investigated, employing high performance liquid chromatography-mass spectrometric method. And by using Caco-2 monolayer model the absorption mechanism of main alkaloids in MEC were studied. Thus, Active components of the medicinal extract of Corydalis yanhusuo responsible of the pharmacological effects were clarified initially by using the vivo and vitro analysis methods. |
