Study On Isolation And Purification Of Immunosuppressive Ingredients Of Medicinal Mushroom Cordyceps Cicadae

In this thesis, the techniques of separation and purification of myriocin from fermentation products of Cordyceps cicadae was studied.C. cicadae, a caterpillar-shaped Chinese traditional medicinal mushroom, is one of the entomopathogenic fungal, which belongs to the class Ascomycetes and DongChongXiaCao group in Chinese herbs.It has been studied by many countries for its characteristic of medicinal value. It is a famous and costly medicinal fungals,and it was investigated clearly in its biological feature and chemical components.Various of effective components such as myriocin, nucleosides, sterols, cyclopeptides, polysaccharides and cordyceps acid are involved in C. cicadae.Now C. cicadae can be produced by unnatural cultivation ,not native collection ,which brings mang advantages of its output and application.Furtermore,its medicinal was studied better,and its applied extent was increased greatly.Therefore,it has significance to explore and develop C. cicadae for national,even international medicine.Myriocin is the most important active immunoregulational component in C. cicadae and it is mostly used in organ transplantation and autoimmune disease. Besides, in C. cicadae anti-fungal and anti-atherosclerosis pharmacological action are also concerned to.The aim of the thesis is to isolation and purification myriocin from fermentation products from C. cicadae,including broth and mycelium, which is based on amplification of submerged fermentation to obtain fermentation products. And then quantitative analysis and qualitative analysis were conducted by a series of analyses. It is proved that the method is simple technology process, and the solvents used are low toxicity and provides the valuable basis for better undersdanding C. cicadae and preparating myriocin for pharmaceutical industry.The methods include NKA macroporous adsorption resin and silica gel (200-300 mesh) which is used as column chromatography, two steps of column chromatograph were used to separate the active component which was screened by antifungal experiment, and then the quantitative analysis and qualitative analysis were conducted by TLC, HPLC, MS and FTIR. Finally, dynamic adsorption model of NKA column chromatography was established.Static adsorption properties of several kinds of macroporous adsorption resin were studied in stage of crude separation. Then, the optimal process conditions for NKA column chromatography were determined after dynamic experiment.Some achieved progresses have been obtained as follows:NKA macroporous adsorption resin was determined after static adsorption properties of several kinds of macroporous adsorption resin were studied. The static experiment data also demonstrated that the adsorption isotherm presented a good fit to Freundlich model, and the thermodynamic results showed that myrioncin adsorption onto NKA was an endothermic process and an entropy-driven spontaneous process. Dynamic adsorption analysis shows that the optimal process condition: concentration of sample 3.0 mg/mL, pH 5.5, flow rate 1.5 BV/h, eluent 60%-70% ethanol solution.Two steps of column chromatography, which is NKA macroporous adsorption resin and sillic gel, were adopted in this thesis. The expetimental results indicated that it is feasible in the process of isolation and purification myriocin from C. cicadae, and myriocin exsited in 60% to 70% ethanol solution. Subsequently, higher purity myriocin was obtained by sillic gel column chromatography. In summary, the taget compound is indeed proved to be a single component by TLC, HPLC, MS, and FTIR.Breakthrough curve model was established on the basis of hypothesis and simfilication by carrying on material balance of the process of myrioncin adsorption onto NKA. The result shows that it was consistent with expetimental breakthrough curve and the overall mass transfer coefficient KFav 0.0188 cm-1 and adsorption band length Za 8.96 cm were obtained.The above results obtained provide forcefully theory and practice guidance, especially provide the valuable basis for better undersdanding C. cicadae and preparation myriocin for pharmaceutical industry. The breakthrough curve model of myrioncin adsorption onto NKA macroporous adsorption resin can gain plenty information. The scale-up rule and scale-up effect of column chromatography would be used widely in the future.