Establishment Of Vitro Culture System Of Tribulus Terrests L. And Investigation Of It’s Secondary Metabolite

Tribulus terrestris L. (T. terrestris), a member of Tribulus genus Zygophyllaceae family, is known as a traditional Chinese herb medicine for its excellent pharmacological activities in preventing and curing many diseases. However, there are still some problems unsolved such as resource limitation and quality instability. At present, the rate of germination and the yield of T. terrestris are low, and the pharmacodynamic effects of T. terrestris from different producing areas varied significantly. Thus, it is of great theoretical and practical significance to establish a culture system with a stable output of active metabolites and to study the growth and metabolism of T. terrestris. To solve the questions mentioned above, we successfully established a culture system of T. terrestris in vitro and preliminary explored the main secondary metabolites, as well as the metabolic rule of T. terrestris.This dissertation includes three parts as follows:1. The establishment of culture system of T. terrestris in vitro(1) The inducing of callus and plantletEmbryo of T. terrestris as an explant is used to induce callus. We studied the growth characterization of T. terrestris selected the most suitable combination of PGR. Therefore, the culture system of plantlets were established by direct plantlet regeneration in vitro.(2) The establishment of suspension culture systemOn the basis of callus cultural, we established the suspension culture system of T. terrestris. At the same time, the major elements in medium were optimized. The results indicated that the most suitable medium included 35 g/L Sucrose,120 mg/L extra KH2PO4,40 mg/mL Casein Hydrolysate and 300 mg/L Yeast Extract Then, we took a further study on the growth dynamic of cells by suspension culture and measured the changes in biomass, protein content, nutrient consumption and pH in the medium. The results showed that the growth of suspension cells act as a sigmoid curve. Besides, the growth cycles of high and low inoculum density are 12 and 20 respectively, and the nutritions in the medium were fully used.-2. The study on secondary metabolite of suspension cells(1) The identification of secondary metaboliteSaponins and flavonoids were the main active ingredients in T. terrestris. We measured the contant of saponins and flavonoids in T. terrestris cells of different periods and different parts. The results showed that T. terrestris plant producted saponin in all period and in all parts of the plant. However, the growing cells including callus and suspension cells in cultured cell system produced little or no saponin, while other steroidal compounds were produced in all parts. These result suggested that the production of saponin in T. terrestris mainly depended on the level of cell differentiation. On the other hand, the flavonoid was undetectable in T. terrestris cells cultureds in vitro.(2) Primary investigation of the metabolic regulation of saponinsIn order to induce T. terrestris cells to produce saponin, we used a variety of common methods to promot the contant of plant secondary metabolites, including plant hormones and elicitor prepared by endophyte of T. terrestris. At the same time, a co-culture system of T. terrestris cell and its endophyte was established. Although these methods were not effective in producing saponin, the endophyte elicitor regulated the growth of T. terrestris cells, and both co-culture system and ABA make T. terrestris suspension cells brown. These experiments laid a solid foudation for further studies.3. The discovery of coumarin and the study of its bioactivitiesWith our studies going on, fluorescent substance was found in our suspension cells. After property experiments, these materials were isolated and identified. The results suggested that it might be 8-hydroxy-6-methoxy coumarin, which is a new ingredients that hadn’t been reported in T. terrestris. Thus, we took further studies the coumarin in T. terrestris. The results showed that the synthetic positions of coumarin is in the root tips in T. terrestris plant. Meanwhile, it was generated in dedifferented cells. Besides, the production of coumarin in suspension cells and callus is stable, and it would not recession with cell subculture.Next, we detected the activity of the coumarin in T. terrestris by 3T3-L1 adipocyte cells, and the results showed that it inhibited the differentiation of 3T3-L1 adipocyte cell, suggesting its ability of inhibiting adipogenesis and regulating lipid metabolism in vitro.In conclusion, we established a culture system of T. terrestris in vitro to study the characteristics of growth and metabolism and a co-culture system of T. terrestris and endophytic fungi. Under above conditions, we firstly discivered a new ingredient, namely coumarin in T. terrestris and we determined its producing site and period. Besides, we explored the metabolic role of coumarin on adipogenesis, suggesting that T. terrestris worthed a deep investigation in our future work.