| Triterpenoid acids are the main bioactive components from Ganoderma lingzhi, which have a range of pharmacological effects. They possess a variety of bioactivities such as anticancer. However, at present the lower of purity, the expensive price and the shortage of triterpenoid acid products on the market, severely limits the further study of its pharmacology and commercial application. Therefore, it is significance to extract triterpenoid acids from different sources of G. lingzhi and analyze its components. Additionally, the stability of triterpenoid acids has an important effect on their extraction and separation. So far, it hasn’t seen any other related reports except Very few cases. Therefore, it is urgent to strengthen the research in this field.In this dissert, fruiting body and mycelium of G. lingzhi were chosen as materials, and the extraction conditions of triterpenoid acids were optimized respectively. Based on this, a preliminary identification of the components of triterpenoid acids were carried out, then the stability was studied.The main results were taken as follows:1. As for fruiting body, through the contrast experiments, alcohol refluxing extraction without enzyme pretreatment was used as a foundation for the study of efficient extraction of triterpenoid acids. Then single factor experiments, Box-Benhnken design and response surface methodology were applied to further optimization. The key factors of extraction, the ratio of solvent to material (A), extraction time (B) and extraction times (C), were optimized by Box-Benhnken design and response surface methodology, and the regression model which can predict yield of triterpenoid acids was obtained:Y=0.33+0.003125A+0.021B-0.014C+ 0.0005AB-0.019AC-0.005BC-0.044A2-0.040B2-0.032C2。 The experimental results showed that the optimum extraction condition were ratio of solvent to material 25:1, extraction time 2h, extraction times 3 and the yield of triterpenoid acids reached (0.335±.008)mg/g, which was consistent with the maximum predicted value.2. G. lingzi CGMCC5.616 was chosen as starting strain, and a mass of mycelium were obtained by shaking-flask fermentation culture. Under the premise of anhydrous ethanol refluxing extraction, the extraction conditions such as ratio of solvent to material, extraction time and extraction times were investigated through single factor experiments. Optimized by central composite rotatable design and response surface methodology, the regression models related to three key factors which can predict the yield of triterpenoid acids was obtained:Y=0.52+0.022A+0.027B+0.064C+ 0.021AB-0.013AC+0.010BC-0.056A2-0.094B2-0.057C2. The optimum extraction condition were ratio of solvent to material 12:1, extraction time 2h, extraction times 3 and the yield of triterpenoid acids reached (0.537±0.005) mg/g, which was consistent with the maximum predicted value.3. ADS-8 macroporous resin was chosen as medium to separate triterpenoid acid. The article examined the effect of elution velocity to triterpenoid acid from G. lingzhi mycelium. It showed that the compositions of triterpenoid acids were different, especially the chromatographic peak for which the retention time is 44.66 min were different in the content; Compared with 1 mL/min and 2 mL/min, two new chromatogram peak (retention time were 66.27 min and 66.27 min) appeared in the flow rate of 3 mL/min. And the chromatographic peaks (retention time is 44.66 min) were different on the content. In addition, the concentrations of ethanol have significant effect on the elution of triterpenoid acid. Triterpenoid acids can be eluted completely with 70%～90%ethanol, most of them were eluted with 60% ethanol, and only a small amount were eluted with 20%～40% ethanol. Further analysis of HPLC showed that there is a significant difference in the components of triterpenoid acid in the mycelia and fruit body, and peaks of triterpenoid acids were relatively late, and rich in components.4. The LC-ESI-MS was used to analyze the constituents of triterpenoid acid from G. lingzhi mycelium. The result showed that it contains a small amount of ganoderic acid A and ganoderic acid B. In addition, there are two major elution peak in mycelium, one is an analogue of ganoderic acid A, the other is the analogue of ganolucidic acid B, but the structure remains to be determined.5. The result showed that the stability of triterpenoid acid from mycelium and fruit body were affected by acid catalyst, and has nothing to do with the polarity of the solvent. The temperature experimental showed that triterpenoid acid from fruit body and mycelium are stable relatively, when the temperature is 25℃ and below. With the increase of temperature, two types of triterpenoid acids degraded in a different degree. |
PDF Full Text Request