Tissue And Cell Culture And Its Synthetic Regulation Of Indole Alkaloids Of Catharanthus Roseus

Catharanthus roseus (L.) G. Don, Madagascar periwinkle, is one of the most extensively investigated medicinal plants. More than 100 alkaloids have been isolated from the different parts of the plant many of them possessing remarkable pharmacological activity. The most important of these are the antileukemic alkaloids vinblastine and vincristine, the antihypertensive alkaloid, especially vinrelbine, a better anticancer drug of indole alkaloids in use, ajmalicine , serpentine, and catharanthine, the precursor of semisynthesis of vinblastine. The low yields of the antileukemic alkaloids in the plant, combined with their high market price has encouraged intense research for alternative methods for the production of these alkaloids, especially, such as synthesis or semisynthesis, cell and tissue culture. Plant cell and tissue cultures have been investigated as an alternate source of production. This thesis is trying to explore the possibility.A reversed -phase HPLC method is established for the determination of tryptamine, serpentine, vindoline, ajmalicine and cathananthine in Catharanthus roseus tissue and cell cultures. These indole alkaloids were successfully seperated on a Nucleosil 5 C18 column (250×4.6mm, 5μm, Phenamenex, USA) with a mobile phase of methanol—acetonitrile—0.025mol/Lammonium acetate— triethylamine (15:40:45:0.1) monitored by a UV detector at 280nm. Intra- and inter- day precisions of these alkaloids were lower than 2% and 3% (RSD). The simple, rapid and sensitive method with good precision was proved by DAD analysis according to UV spectrum of these alkaloids in standards and samples.In this experiment, callus can be initiated in different medium MS, B₅ or 6,7-V with hormones combination of 2,4-D, NAA and 6-BAP, KT, almost all leaves can be induced into callus. Six callus lines were established from different plants of Catharanthus roseus , of them, P2,P5.CR4C have relatively higher ability of alkaloids synthesis, Nutrients and culture factors influencing accumulation of alkaloids were investigated, phosphate ,nitrogen, sugar and illumination greatly influence the callus growth and alkaloids accumulation.During Leaf callus initiating in four different media under illumination or in dark. The content of vindoline and cahtaranthine declined rapidly, while ajmalicine and serpentine increased markedly; Activities of acidic and alkaline peroxidase increased in tendency. Results also showed that illumination improved occurrence of leaf callus. and stimulated peroxdase activity .The increasing of peroxidase activity correlated with increasing of serpentine contents, 2,4-D repress biosynthesis of indole alkaloids and peroxidase activities in all cultures.Suspension cell cultures were established directly from leaf and stem of...