Study On The Extraction Of Colchicines From Day-lily And Its Bacteriostasis

Colchicine, also known as organic amines alkaloid,due to bond with cells tubulin sub-unit specifically and prevent the formation of microtubule protein, resulting in the structure of the spindle can’t be formed, and inhibition of cell division, can be used as anticancer drugs in medicine,and be used for cultivation of polyploid plants in agriculture also. However, the distribution of colchicine alkaloids in nature is very narrow, and its content is low, can’t meet needs of the society in addition the source is limited. This thesis firstly studied three methods’ parameters of extraction of colchicine from day lily, the supercritical fluid extraction, the ultrasonic assisted extraction and oscillation impregnated extraction. Then, investigated the parameters of purification and separation of unpurified extractive with column chromatography form the dynamic and static angle. Finally, roughly discussed the inhibition performance of colchicine on the growth of E.coli and S.aureus.1.Extraction of colchicine. Firstly,systematically studied the extractive parameters of supercritical carbon dioxide fluid extraction extracted colchicine from day lily, mainly focused on the effects of extraction pressure, time, temperature, and the type, dosages, concentration of the entrainer these factors on the extraction efficiency. The results show that, within the selected range of experimental parameters, the influencing order in which various factors as follows:the most significant effect on the results was extraction temperature, then the entrainer volume, extraction time and pressure in turn ranked subsequently, and the best extraction parameters combination is:15MPa,1.5h,41℃,200mL.Secondly, researched the extractive parameters of ultrasonic assisted extraction extracted colchicine from day lily, mainly discussed the impact of these factors of temperature, time, type of solvent and concentration, solid-to-liquid ratio on the extracted quality. The results show that, within the selected range of experimental parameters, the influencing order in which various factors as follows: the most significant effect on the results was Solid-liquid ratio,then the extraction time, extraction temperature and raw material particle size were ranked subsequently,and the best extraction parameters combination is:1.5h,25 ℃,100mesh,1:35. Finally, discussed the extractive parameters of oscillation impregnated extraction extracted colchicine from day lily, mainly studied the effects of factors such as temperature, time, shaking speed, type of solvent and concentration on the extracted result. The results show that, within the selected range of experimental parameters, the influencing order in which various factors as follows: the most significant effect on the results was Solvent concentration,then the extraction time, the raw material particle size and the oscillation speed turn ranked subsequently. The optimum combination of conditions is:the most significant effect on the results was solvent concentration, then solid-liquid ratio, oscillation speed and extraction time in turn ranked subsequently.2.Purification of the unpurified extractive This paper researched the conditions of column chromatography purification of unpurified extractive from the dynamic and static points of view. First of all, tested the size of the saturated adsorption amount of the selected resin. Secondly, discussed the effects of the eluent volume, concentration, pH value on the effects of the static adsorption and desorption. Then, discussed the effect of three kinds of macro porous resins with different combination modes on the purification and separation of colchicines, and mainly focused on the effects of the concentration volume, pH value of eluent and eluotropic rate on dynamic effects of the adsorption and desorption. The results show that, the order of the saturated adsorption amount of resin is:AB-8>X-5> NKA-9; the purification ability of Resin with different combination way from strong and to weak is:step by step, blending, series; and the optimum combination conditions of static desorption is: desorption time of4.0h, the pH value of10.0±0.02, a concentration of95%ethanol50mL;the optimum combination conditions of dynamic desorption is:pH of6.87±0.02, a concentration of85%ethanol70mL and the flow rate is1.6mL/min. The purity of extractive could reach the point of91.20%after purification.3.Determination of molecular weight of Colchicine and of it’s antibacterial property study In this paper, the molecular weight of purified colchicine were determined, and studied the performance of colchicine for inhibition of E. coli and S. aureus growth. First, added different concentration of colchicine, to the culture medium, and then explored antibacterial properties of colchicine by the determination of bacterial growth curve. On this foundation, further verified this antibacterial properties by spread plate method and streak cultivation. Experiment results show that, the molecular weight of colchicine is400.2; Colchicine could inhibit the growth of E. coli and S. aureus, and this inhibition enhanced with increase of Colchicine concentration; And the antibacterial property is more obvious for the growth of E. coli then for S. aureus at the same concentrations.