| Ganoderic acids (GAs) were one of main bioactive components produced by Ganoderma lucidum, a famous medicinal fungus. They possess a variety of bioactivities such as anticancer. However, pharmacological researches and commercial application of GAs is being severely limited by the unavailability of sufficient mass. The two-stage culture process by combining liquid shaking culture with static culture developed by our lab is an efficient method for the production of GAs. Nevertheless, lack of efficient preparation techniques and related theory still hinders their further clinical research and development. Additionally, the stability of GAs has an important effect on their separation and analysis. So far, only our lab reported the stability of7-O-ethyl-GA-O, the stability of other GAs was still remained unclear. In this dissertation, the GAs from G. lucidum were purified and identified. Their stability was subsequently explored. Based on that information, a novel method was developed for the simultaneous preparation of four GAs. Furthermore, a low-pressure liquid chromatography methodology and a general rate model were established for the separation of GA-T.In this dissertation, by employing silica gel column chromatography, thin layer chromatography and semi preparative high performance liquid chromatography, eight GAs were separated and purified. They were identified as ganoderic acid Mc, DH,7-ethyl-O-GA O (Type Ⅰ), Mk, S, T, Mf and R (Type Ⅱ) by UV, IR, MS and NMR techniques. Among them, GA-DH was a new GA. Subsequently, the stability of the two types of GAs was studied and the results showed that Type Ⅰ GAs were stable in aproptic solvents, while not in proptic sovents. Owing to the stable conjugated system and uniform distribution of electron cloud, Type Ⅱ GAs were stable in both types of solvents. Based on these results, an RP-HPLC-PAD was developed for the simultaneous determination of those two types of GAs. The HPLC operating conditions were optimized and the chromatographic separation was performed on a C18column with a gradient acetonitrile-water as mobile phase. With the good linear relationship, precision, accuracy and recovery, the method could be a reliable analytical platform for the separation and investigation of biosynthesis and regulation of GAs.Pre-separation has significant impact on the subsequent purification. Traditional pre-separation method for GA preparation is tedious and inefficient. In this part, using GA-Mk and-T as model molecules, we explore the performance of MARs on the separation of GAs. By static experiments, ADS-8was selected as ideal separation media among the six resins. The results of kinetics experiments showed that the adsorption data could be well fitted by the peso-second order model. Thermodynamic analysis indicated the exothermic and spontaneous nature of the adsorption process. Dynamic adsorption tests were performed on an ADS-8resin-packed column to obtain optimal parameters for recovering GA-Mk and-T from G. lucidum extract. Under optimized conditions, a laboratory scale-up preparation of GA-Mk and GA-T was carried out. The contents of GA-Mk and GA-T were increased from4.5and2.2%in the crude extract to34.1and14.5%in the final product with recovery yields of90.1and72.2%, respectively. These results demonstrated that ADS-8resin chromatography could act as a useful approach for obtaining ganoderic acids from G. lucidum.Based on the above results, a new extraction/hydrolysis method using50%aqueous ethanol system containing50mmol/1HCl as extractant was developed for the recovery of four type Ⅱ GAs (GA-Mk, T, S and R) from the mycelia of G. lucidum. This simple and integrated process did not efficiently enhance the yields of type Ⅱ GAs by conversion of type I GAs, but also considerably improve the subsequent purification. This one-pot extraction/hydrolysis process increased the yield of the four GAs to200-400%, compared to a raw sample without hydrolysis. In addition, the use of aqueous ethanol as extactant for GAs extraction was important, because it allows that without any conditioning step, the GAs could be recovered by macroporous adsorption resin. Hence, the drawbacks of traditional separation method of GAs were eliminated by using the current method.Currently, the final purification of GA-T was almost by HPLC, which is costly and difficult for scale-up. In addition, the related chromatographic theory was a void. Herein, the performance of low-pressure LC for the preparative separation of GAs was studied. The result that showed highly efficient GA-T preparation can be achieved by low-preesure LC. Its purity and recovery were all over90%. For the first time, gram scale preparation of GA-T was achieved on the column with dimension42×273mm in one run, with chromatographic time less2hours. Thses results clearly demonstrate that could take the place of HPLC for the preparation of GA-T. Subsequently, based on the data from low-pressure LC, a general model was established for the characterization of the separation of GA-T on low-pressure LC. The results showed the calculated profile was well fitted to the eluton curve of GA-T, which suggested the established model could well characterize the chromatographic separation process. Further study on the parameters of the model revealed that the model was not sensitive to the Biot number, while sensitive to the Pe number when it was less1000. The tortuosity factor and bed void fraction εb has a significant influence on the calculated resulted. Among them,εb only easily change the retention time of chromatographic peak, but also affect ipeak width. The developed model could be used for the separation optimization and scale-up of GA-T and other GAs.Taken together, in this work eight GAs were separated and identified from G. lucidum. Their stability was studied and simultaneous quantitative analysis method was developed. Based on these results, the efficient preparation method of individual GA and related chromatographic model were developed, which it is making the sufficient supply of high-purity GA required for its pharmacological studies possible in the future. Moreover, this work could be also helpful for the preparation of other natural products. |
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