Study On Alkaloid Biosynthesis In Cell Cultures Of Corydalis Saxicola Bunting

Plant cell culture is regarded as a promising way to provide an attractive alternative source for limited natural resources. The objective of this work was to establish the cell cultures of Corydalis saxicola Bunting for production of active alkaloids. This work mainly focused on the establishment of cell cultures, elicitor-stimulated alkaloid biosynthesis and pharmacological effects of alkaloids. The main results were shown as follows:1. Callus induction and cell suspension cultures were established through selection of explants, medium and conditions of cultivation. Leaves of C. saxicola were the most suitable explants and modified B5 medium supplemented with 0.5 mg/l 2,4-D plus 2 mg/l BA was the most favorable medium for callus formation. An inoculum size of 7.5-10% (FW) was suitable for cell growth and small inoculum size could impress biomass accumulation. The B5 and MS basal medium were fit to cell growth and the most favorable initial pH value of medium was 6.0. The growth period of cell cultures was around 24 d with highest biomass on day 18.2. An effective spectrophotography was established to determine the total alkaloids of C. saxicola. Results showed that alkaloid content in cells was much lower than that in plant. Two different methods were used to extract total alkaloid in cultured cells. But there was no significant difference in these two methods, which were carried out on their optimal conditions, respectively. Alkaloids in cultured cells were identified by the method of TLC, LC-MS et al. The main alkaloids in cells were dehydrocavidine and berberine. And there may be some new alkaloids in cells. A RP-HPLC method was established to measure the content of dehydrocavidine and berberine. It was a reliable method with good precision. The content of dehydrocavidine in cells was lower than that in plant. However, berberine was undetectable in plant.3. The cell growth of suspension cultures and metabolic process of nutrition compounds in medium were investigated in 80 ml flask. It was showed that the pH value of suspension cultures fluctuated between 5.0 and 7.0. The growth curve of C. saxicola cells appeared as a"S"shape. Phosphate in medium was ingested quickly, followed by NH4+, C and NO3-. During scale up cultures in flask, it was found that cells grew most quickly in 100-ml medium, and produced the highest biomass.4. Fungal elicitor obtained from cell wall of Aspergillus niger could improve the production of alkaloid in suspension cell cultures. The elicitor concentration of 50 mg/l was the most favorable for cell growth and alkaloid production. Furthermore, the treatment of fungal elicitor stimulated the production of active oxygen species, improved the activities of phenylalanine ammonia lyase (PAL), resulted in cell death and enhanced the production of soluble proteins and secondary metabolites. The results suggested that elicitor-induced defense response initiated the alkaloid biosynthesis. Moreover, the relationships among Ca²⁺, H2O2 signals, jasmonic acid (JA) and alkaloid biosynthesis were investigated in suspension cultures of C. saxicola. The results showed that elicitor-induced alkaloid biosynthesis was mediated by Ca²⁺ signals to generate defense response in C. saxicola cells. Generation of H2O2 associated with elicitor treatment leaded to lipid peroxidation, but it could not contribute to alkaloid production. Cells treated with fungal elicitor resulted in the increase of jasmonic acid content, which leaded to the biosynthesis of alkaloids. However, addition of inhibitors of lipoxygenase could not completely impress the increase of alkaloid production induced by fungal elicitor. The obtained results indicate that synthesis of jasmonic acid associated with fungal elicitor seems to be only part of a complex regulatory mechanism for the onset of many, but not all, defense reactions including alkaloid production in C. saxicola cells.5. Alkaloid extraction of cells showed more protective effects on acute and chronic liver injury of mice by CCl₄ than that of plant. The cell extraction could decrease levels of ALT, AST and MDA, which increased siginificantly in mice induced by CCl₄, and increase activities of SOD in mice treated with CCl₄. The results suggested that this protective effect of cell extraction was related to cleaning free radical and preventing lipid peroxidation.