| No complicated synthetic reaction is involved in the biotransformation reaction of medicinal plants, so it saves time and money. However, the biotransformation reaction of medicinal plants started relatively late and is mostly confined to laboratory, far from satisfying the market requirement. In order to further provide useful data for the biosynthesis of Alkaloid, this thesis carries out a series of experiments on Trichosanthes kirilowii suspension cells and provides experiment evidence for industrial production. The study is mainly focused on the following aspects:Ⅰ. Induction of Trichosanthes kirilowii CallusThe wild Trichosanthes kirilowii stems are used as the explant, and aseptic seedling is obtained after induction. The tender stems of the aseptic seedling are used as the explant, and the callus is induced under two different culturing conditions. The results indicate Y2:MS+6-BA0.5mg/L+NAA0.2mg/L+30g/L sugar. With the highest inductivity, callus is loose and is in the shape of particle, and the cell homogeneity is good.Ⅱ. Induction of Trichosanthes Kirilowii Suspension Cells and the Optimization of the Culturing ConditionTransform the Trichosanthes kirilowii callus after many times subculture into suspension cells subculture. After many times of subculture, screen the suspension cell system in good state. The growth condition of the suspension cells is optimized. The experiment condition most favorable to the growth of Trichosanthes kirilowii suspension cells is as follows:initial inoculum size1.5g/60ml, rotating speed105r/min, temperature(26±1)℃, pH value5.8～6.2.Ⅲ. Trichosanthes Kirilowii Suspension Cells’ Biotransformation of Exogenous SubstratesA variety of lead compound is biotransformed by means of Trichosanthes kirilowii suspension cells systems. After a series of process such as extraction, silica gel separation, high performance liquid, the transformed product is separated and purified. Then, we define its molecular weight by high resolution mass spectrum, and further the molecular formula and degree of unsaturation. And its structure is determined further by NMR spectroscopy. By1H-NMR、13C-NMR、DEPT, the category and number of substituent group can be defined; and by HSQC spectrum, every hydrogen signal and carbon signals is attributed to certain groups; and by HMBC and1H-1HCOSY spectrum, the location of substituent group can be defined; and finally by coupling constant and NOESY spectrum, the three-dimensional configuration of the compound is defined. This experiment is the first one to focus on biotransformation of Trichosanthes kirilowii suspension cells in which two new compounds are obtained, blazing a way in the Trichosanthes kirilowii suspension cells.IV. Content Measurement of Asiatic Acid in Trichosanthes Kirilowii Suspension Cells SystemFirst, create the method for measuring the content of asiatic acid in Trichosanthes kirilowii culture system-hromato graphic column:Eclipse Plsus C18(4.6mm*250mm,5ug);mobile phase:methyl alcohol-water(65:35); flow rate:lml\min; column temperature:30℃; measurement wavelength:210nm; linear equation y=922.85x-5.7471; correlation coefficientR2=0.9991; This method boasts of features of simple operation, good repeatability, qualified recovery rate, accurate measurement result, providing important evidence for the subsequent experiments.V. Condition Screening for the Biotransformation of Asiatic Acid by Means of Trichosanthes Kirilowii Suspension Cells Culture SystemThe single factor examination of Trichosanthes kirilowii suspension cells transformation system, transformation time, substrate input and cell density, plus the experiments on the enzyme transformation in the Trichosanthes kirilowii suspension cells system all boil down to the conclusion that the enzyme system of higher plants also have the ability to be transformed, which lays the important experimental foundation for industrial production.VI. Impact of Trichosanthes Kirilowii Suspension Cells Transformed Product on Multiplication of Three Kinds of Cancer CellsWe examine the effect of the two kinds of new compounds on the inhibition ratio of cell multiplication in breast cancer cell strain MDA cell, SW480(colorectal cancer cell), Hela (cervical cancer cell) by means of MTT method.VII. Preliminary Regulation of Trichosanthes Kirilowii Suspension Cells SystemWe carry out study on the regulation of biotransformation of Trichosanthes kirilowii suspension cells system by sodium pyruvate, pyroracemic acid, a-pinene, squalene, as well as on the impact of the four substances on the vitality of peroxidase, catalase and polyphenol oxidase in the transformation system. The results indicate that precursor substance exerts certain effects on transformation rate, and remarkable effects on the activity of those three kinds of enzymes. The precursor substance has certain impact on the cell toxicity, oxygen consumption and cell browning, thus influencing biotransformation. |
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