| Dioscorea zingiberensis C. H. Wright belongs to Dioscoreaceae. Its tuber is consumed as a food and used as a Traditional Chinese Medicine(TCM) for long time. In China, Dioscorea zingiberensis is widely distributed in central China, such as Shanxi, Hubei and Hunan provinces, and formed a new kind of agricultural industry. The reason for large-scale planting on agriculture is that Dioscorea zingiberensis C. H. Wright has high content of saponins and diosgenin. These nature product have good effect on treatment of cardiovascular disease, enhance immunity, antibacterial and antitumor. Diosgenin((25R)-5-Spirosten-3β-ol) is main structural skeleton of saponins. It is one of the most important starting material for the semisynthesis pharmaceutical industry of steroidal drugs. And it also has some significant biological activities. Diosgenin production industry brings huge economic benefits to the local people and provides an important synthetic raw material to downstream firms. However, the traditional method of diosgenin production is inorganic acid hydrolysis method. Dioscorea zingiberensis C. H. Wright tubers are hydrolyzed by hydrochloric acid or sulfuric acid to produce diosgenin, which discharges low pH wastewater into environment. This has caused serious environmental pollution. The traditional extraction method of total steroid saponins is heating stirring extraction, which is low extraction rate and low efficiency. In order to solve above two problems, this study designed a simple and effective steroidal saponins extraction process according to the properties of steroidal saponins an d diosgenin. On this basis, the "green solvents" acidic ionic liquid was used in catalytic hydrolysis experiment to produce diosgenin. And content and activity evaluation of saponins and diosgenin were investigated. The main results were as follows:1. The design and optimization of the new extraction processThree kinds of extraction process were designed and optimized. Ultrasound-microwave assisted extraction technology was used to extract total steroidal saponins. The single factor experiments were carried and the optimum extraction condition were as follows: ethanol concentration 70%, solid-liquid ratio 1:15 g m L-1, microwave power 500 W and extraction time 420 s. Under the optimum extraction condition, the extraction yield represented with diosgenin was 1.35%. Then ionic liquid based ultrasonic-microwave assisted extraction of steroidal saponins was employed. The single factor experiments were also carried and the optimum extraction condition were as follows: extract solvent [EMIm]BF4, [EMIm]BF4 concentration 0.5 mol L-1, solid-liquid ratio 1:15 g mL-1, microwave power 500 W and extraction time 480 s. Under the optimum extraction condition, the extraction yield represented with diosgenin was 1.02%. Enzyme-based ultrasonic-microwave assisted extraction was used and single factor experiments results were complex enzyme(cellulase and pectinase, 1:1, w/w), enzyme buffer pH 5.0, enzyme concentration 0.8%(w/w), ethanol concentration 70%, solid-liquid ratio 1:15 g m L-1, microwave power 500 W and extraction time 300 s. Then the Box-Behnken response surface was used to optimize the extraction process. The optimum condition were enzyme buffer pH 5.2, ethanol concentration 73.7% and microwave power 547.9 W. The corresponding predicted response values under these conditions was 1.46%. Then the verification experiments were performed under the optimized conditions in three replicates and the actual result was 1.41%, which were largely consistent with the predicted value. This good correlation indicated that the models were statistically reliable and can be used to optimize the process of steroid saponins extraction from Dioscorea Zingiberensis C. H. Wright. At last, scanning electron microscope(SEM) was used to observe the influence of different processing methods on the plant cell.2. The new preparation method and hydrolysis mechanism researchAcidic ionic liquid is a kind of acid catalyst, which has a broad prospect because of its outstanding advantages, such as stable chemical state, recycled use and high catalytic efficiency features in catalytic hydrolysis steroidal saponins. In this study, firstly [EMIm]HSO4 was used to catalytic hydrolyze steroidal saponins to diosgenin. After the response surface optimization, 29.88 ± 0.45 mg diosgenin was obtained from 0.3 g saponins under [EMIm]HSO4 concentration 8.5 mol L-1, reaction temperature 100℃, solid-liquid ratio 1:22 g mL-1 and reaction time 7.3 h. Then functionalized acidic ionic liquid [BHSO3MIm]HSO4 was employed and compared with [EMIm]HSO4. The [BHSO3MIm]HSO4 reaction condition were concentration 5 mol L-1, reaction temperature 100℃, solid-liquid ratio 1:15 g m L-1 and reaction time 5 h. Then Microwave heating method was introduced to the experiment. The new method condition were [BHSO3MIm]HSO4 concentration 4 mol L-1, reaction temperature 100℃, solid-liquid ratio 1:15 g m L-1 and reaction time 20 min. Under these condition 29.97 ± 0.51 mg diosgenin was obtained from 0.3 g saponins. In addition, [EMIm]HSO4 and [BHSO3MIm]HSO4 were compared and the result showed that acidity of ionic liquid was a very important parameter. The pathways of hydrolytic process were studied. This process is summarized into three stages: long sugar chain saponins were hydrolyzed into secondary saponins with short sugar side chain firstly. Then these secondary saponins were hydrolysed into shorter side chain saponins. Finally, sugar side chains of saponins were hydrolyzed completely to target product diosgenin.3. The preparation and application of immobilized ionic liquidThe immobilized acidic ionic liquid was prepared by sol-gel method. Immobilized acidic ionic liquid was made from ethyl silicate and [BHSO3MIm]HSO4. Then it was characterized by infrared spectrum and scanning electron microscopy(SEM). The results showed that the immobilized ionic liquid was prepared successfully. The immobilized ionic liquid was used in hydrolysis experiments. According to experiment condition, optimum experiment method was as follows: 0.1 g saponins were fully dissolved in 70% ethanol solution, then 2 g immobilized ionic liquid was added. The reaction was carried for 4 h at 100℃. After the reaction, 20 times of water was added to the reaction system in order to dilute alcohol. Diosgenin was extracted and detected.4. Characterization of diosgenin products and separation of starchAppearance analysis, scanning electron microscopy(SEM) analysis, infrared spectrum analysis and HPLC analysis were carried. The result showed that the purity of diosgenin product was the 92.3% and has good quality.In order to take full advantage of plant resources, two kinds of starch separation process were designed. One process: Fresh Dioscorea zingiberensis C. H. Wright(Dry Dioscorea zingiberensis C. H. Wright was soaked in water for 24 h) were grinded with water. The serous fluid was washed by water, then filtrate was collected for stratification. Starch was at the bottom. The other process: Dry Dioscorea zingiberensis C. H. Wright powder was used to extract total saponins. Then extraction residue was treated by cellulase. At last, the crude starch was collected.5. Inhibition of bacterial biofilm activity and the anti-cancer activityInhibition of bacterial biofilm activity of typical steroidal saponins was investigated. Inhibition activity of dioscin, gracillin and protodioscin increased with the increase of concentration. Inhibition rates were greater than 80% at the concentration 4 mg mL-1.The anti-cancer activity of dioscin, gracillin and protodioscin was investigated by cell apoptosis experiments. The three kinds of typical steroidal saponins had good inhibitory effect to the lung cancer cells, which showed obvious time and dose dependent. Their effect was better than positive control 10-Hydroxycamptothecin(10- HCPT) when concentration was 10 μg m L-1. |
PDF Full Text Request