Analysis Of Proteins In Three Fungi Traditonal Chinese Medicines By Gel Electrophoresis

Ganoderma lucidum(GL), Cordyceps sinensis(CS) and Morchella volgaris(MV) are three famous traditional Chinese medicines(TCMs) that had long been used for the treatment of various diseases in China. Proteins, which have lots of biological activities, are one of the main chemical compositions in those three fungi medicines. Although only few studies about the analysis of proteins in those three medicines had been reported, protein itself is expected to become a chemical marker for the quality control of TCMs. On the other hand, SDS-PAGE and two-dimensional electrophoresis(2-DE) are the key techniques in proteomic study. Therefore, in this thesis, three fungi medicines(GL, CS and MV) from different production areas of China are differentiated and evaluated based on the protein components analyzed by SDS-PAGE and 2-DE. There are six main parts(chapters) in this thesis.In the first chapter, the pharmacological activities and analytical methods for plant origin proteins were summarized. Then, three fungi TCMs include GL, CS and MV were briefly introduced.The second chapter was focused on the analysis of proteins in GL from 14 different producing areas. The proteins in GL samples were successively analyzed by SDS-PAGE, IEF-PAGE and 2-DE. SDS-PAGE results showed that the molecular weights of proteins in GL samples were from 15.0-97.2 kDa, and the numbers and abundance of protein bands differed greatly among 14 samples. Furthermore, the IEF-PAGE results showed that the main p I values of proteins were 4.0-7.5. In addition, the 2-DE results further indicated that the numbers and abundance of protein spots of GL samples from 14 different producing areas were varied greatly. Those results can provide a reference for further differentiating and evaluating of GL samples.In the third chapter, protein components in natural CS samples from 26 different production areas were analyzed by SDS-PAGE, IEF-PAGE and 2-DE. And the crude proteins fractionated by PEG(sample preparation purpose) were also investigated. The SDS-PAGE results showed that the numbers and optical density curves of protein bands of CS samples from 26 different production areas were very similar, and the molecular weights were 6.5-97.2 kDa. And the pI values of proteins were 4-7 in IEF-PAGE profiles. On the other hand, the numbers of protein spots were not obviously increased by PEG fractionation and the experimental procedure was relatively complicated, so the total proteins were analyzed by 2-DE. The results of total proteins' 2-DE profiles showed that the numbers and abundance of protein spots existed difference in 26 samples. Then based on matched protein spots and their expression values, 26 CS samples from different producing areas were mainly distributed into two categories(Tibet and Qinghai) in hierarchical analysis.The fourth chapter was focused on the analysis of proteins in MV from 7 different producing areas by SDS-PAGE, IEF-PAGE and 2-DE. The results of SDS-PAGE(protein samples obtained by TCA/acetone) showed that the numbers and optical density curves of proteins were similar in 7 MV samples, and p I values were 4-7 on IEF-PAGE protein profiles. The 2-DE results also showed that the numbers and abundance of protein spots differed in 7 samples. The results of this chapter can provide a reference for identification and evaluating of MV samples.In the fifth part, 2-DE combined with MALDI-TOF-MS/MS was applied to study the antiplatelet mechanism of Rhizoma Corydalis(RC). The results showed that total 52 differentially expressed protein spots(more than 1.8 fold) were found on the 2-DE protein profiles, and 51 of them were identified by MALDI-TOF-MS/MS. Those proteins were directly related with cytoskeleton, Ca2+, and platelet activation signal pathways. The results showed that P2Y12 receptor might be one of the direct target proteins of RC in platelet to mediate G?i signaling pathway for inhibition platelet aggregation function.The sixth part is conclusion and prospective of the present study. The SDS-PAGE and 2-DE were applied for the analysis of proteins in three fungi medicines from different producing areas. This thesis can provide a reference for the quality evaluation of those three medicines, and also enrich the chemical makers for the quality control studies of TCMs. In addition, study on the antiplatelet aggregation function of RC can provide a basis for further investigating on the antiplatelet aggregation signal pathways of active components in RC.