The Establishment Of Suspension Cell Cultures Of Gynura Divaricata[(L.) DC.] And The Analysis Of Main Components

Gynura divaricata [(L.) DC.] belongs to Gynura Compositae and is a kind of goodmedicine and food plants, which is widely used in the treatment of hypertension,hyperlipisemia and diabetes and so on. But the plants contain a kind of hepatotoxicpyrrolizidine alkaloids (HPAs) which makes the utilization of the plants restricted. Inaddition, the cultivation of Gynura divaricata [(L.) DC.] is restrained by ecologicalenvironment, natural environment and arable area. It is a new way to explore Gynuradivaricata [(L.) DC.] resources by plant tissue culture technology to remove HPAs andaccumulate effective ingredients.The thesis discussed the induction of callus, the establishment of suspension cellculture system overall and the reduction of toxic alkaloids through the plant tissue culturetechnology. Besides, the thesis compared the content of physical and chemical components,the active ingredient content among dried leaves, callus and suspension cell. The resultsshowed as follows:1. This chapter mainly studied how to induce and screen embryonic callus from explants,induction period, basic medium, hormone concentration and the ratio. The results showed:Leaves with buds of Gynura divaricata can be fast induced callus on MS+1.0mg/L KT+0.5mg/L2,4-D+0.3mg/L NAA+1.0mg/L6-BA culture medium and the best time forinduced treatment was in the3rdmonth. Gynura divaricata can be received crisp vigorouscallus with metal luster. The growth curve was shaped “S”, and the highest biomassreached19.54g/bottle yielded at18thday after inoculation.2. This chapter established stable suspension cell cultures of Gynura divaricata. Thefactors on culture generation, initial cultural density, volume amount, initial pH value,hormone types and concentration were optimized. The results showed: The most suitableconditions of cell suspension cultures were found to be B5medium supplemented with0.5mg/L NAA+2.0mg/L6-BA+30g/L sucrose, pH7.0,40%volume amount,90g/L(freshweight) initial cultural density, culture temperature(25±2)℃, shaking at115r/min withweak light training. Suspension cell growth curves were like “S”. The pH value on culturedmediums were reduced at first, and then increased gradually. The change of cell vitalitywas reduced gradually lagging that of cell growth curves.3. This chapter mainly used the physical and chemical method and HPLC/MS/DADdetection methods to analysis the content of toxic component, conventional component andantioxidant activity compounds among Gynura divaricata dried leaves, callus andsuspension cell. The results showed: Gynura divaricata dried leaves were measured11.73μg/g retrorsine under the HPLC lab condition, however, callus and suspension cellcouldn’t detect the alkaloid. The content of ash, lipid, total triterpenoid in Gynura divaricata dried leaves were higher than callus and suspension cells. The coarasepolysaccharide content was highest in suspension cell and least in dried leaves. The contentof total flavonoids was callus> leaf> suspension cell. Gynura divaricata antioxidantactivity active ingredient was mainly dissolved in methanol solvent and identified twotypes of active substances for chlorogenic acid and dicaffeoylquinic acid byHPLC/MS/DAD. The size of amount was callus> suspension cell> leaf, and the contentof CHA and DCQA in callus were0.85and1.7mg/g.