基于加压双相酸水解技术萃取薯蓣皂苷元的研究

发布时间：2018-05-06 21:49

本文选题：薯蓣皂苷元 + 薯蓣皂苷　；参考：《江苏大学》2017年硕士论文

【摘要】：薯蓣皂苷元在药物生产中是一种非常重要的前体化合物,是合成避孕药、可的松、性激素、蛋白同化激素等甾体类药物的主要原料,因此在医药界有“药用黄金”和“激素之母”的美誉。此外,药理研究表明,薯蓣皂苷元具有多种生物活性,包括抗癌、抗炎、提高免疫力、抗血栓、降血脂和抗病毒等。在我国,黄姜和穿山龙因薯蓣皂苷元的含量较高,是生产该化合物的主要原材料。植物中的薯蓣皂苷元主要以皂苷形式存在,因而生产薯蓣皂苷元必须使糖苷键水解。传统方法生产薯蓣皂苷元导致了严重的环境污染,非皂苷部分得不到利用,而且薯蓣皂苷元收率较低。本论文研究旨在建立一种更清洁和高效地从黄姜和穿山龙中生产薯蓣皂苷元的方法,并提高药用薯蓣资源的利用效率,已完成的研究工作主要包括:(1)建立了HPLC-UV测定薯蓣皂苷元含量的方法建立了HPLC色谱分析条件,即色谱柱为Waters XBridge Shield RP18(4.6mm×150 mm,5μm);流动相为乙腈:水(75:25);流速为1.0 mL/min;检测波长为203 nm;进样量为20μL;柱温为40oC;分析时间为15 mins。在该条件下,薯蓣皂苷元可以与干扰成分实现基线分离(R1.5)。此后,进行了方法学考察,包括线性关系、精密度、稳定性、重复性和加样回收率。结果表明,薯蓣皂苷元在0.002~1.024 mg/mL范围内线性关系良好(r2=0.9999);精密度实验RSD=1.02%(n=6);重复性实验RSD=1.78%(n=6);平均加样回收率分别为96.2%(RSD=1.17%)、100.4%(RSD=2.47%)和101.6%(RSD=1.09%);供试品溶液在24 hrs内测定,RSD=1.93%。由此可见,所建立的HPLC分析方法是稳定、可靠的,可用于薯蓣皂苷元的定量分析。(2)直接加压双相酸水解法从黄姜中萃取薯蓣皂苷元首先利用正交实验设计对加压双相酸水解反应条件进行了优化,考察了反应温度、硫酸浓度、反应时间和料液比四个因素对薯蓣皂苷元收率的影响。利用极差分析和方差分析对4个因素的影响进行评价,并得到最佳提取条件。此后,验证了最佳提取条件,并将直接加压双相酸水解法与两种传统方法和预发酵-双相联合酸水解法进行比较。此外,利用硅胶柱色谱和重结晶对双相酸水解后的萃取物中的薯蓣皂苷元进行了分离与纯化,并检测了所得薯蓣皂苷元纯度。结果表明,当反应温度为140oc、硫酸浓度为0.2mol/l、反应时间为1.5hrs、料液比为2.5g/50ml时,薯蓣皂苷元的收率达到2.21%。采用新方法的薯蓣皂苷元收率比两种传统方法和预发酵-双相水解法分别高出63.7%、70.4和20.8%,且所需硫酸浓度仅为传统方法的约1/10。由此可见,利用直接加压双相酸水解法从黄姜中萃取薯蓣皂苷元的收率较高,硫酸用量明显减低,反应时间缩短。(3)总皂苷提取—加压双相酸水解法从穿山龙中萃取薯蓣皂苷元首先以70%乙醇溶液对穿山龙中总皂苷进行索氏提取,再利用加压双相酸水解法水解皂苷产生薯蓣皂苷元。采用单因素实验考察了料液比、硫酸浓度、反应温度和反应时间四个影响因素,并进一步通过正交实验设计对酸水解条件进行优化,得到最佳反应条件,即料液比为8g/50ml、硫酸浓度为4μl/ml、反应温度为150oc、反应时间为2.5hrs。与传统方法相比,采用该方法的薯蓣皂苷元收率为1.67%,提高了66.02%,并且硫酸消耗量为1.497ml/g,降低了97.00%。由此可见,采用总皂苷提取-加压双相酸水解法,能更高效、更清洁地从穿山龙中萃取薯蓣皂苷元;也表明加压双相酸水解是从薯蓣属植物中生产薯蓣皂苷元的一种有效的途径。(4)综合利用—加压双相酸水解法从新鲜黄姜从萃取薯蓣皂苷元首先采用物理法回收新鲜黄姜中的淀粉和纤维素,再将粗皂苷进行加压双相酸水解萃取薯蓣皂苷元。利用单因素实验依次考察了料液比、硫酸浓度、萃取溶剂体积、反应温度、反应时间、转速以及石油醚沸程等因素对薯蓣皂苷元收率的影响,并优化得到最佳反应条件。此后,以薯蓣皂苷元收率、硫酸消耗量以及产生废水中的化学需氧量(cod)和总有机碳量(toc)为评价指标,将新方法和直接加压双相酸水解法以及传统方法进行了比较。结果表明,当料液比为1g/100ml(粗皂苷粉末质量/硫酸溶液体积)、硫酸浓度为2μl/ml、萃取试剂为30ml石油醚(90~120oc)、反应温度为130oc、反应时间为2hrs、搅拌速度为100r/min时,薯蓣皂苷元的收率可达到4.17%。与传统方法相比,薯蓣皂苷元收率提高了22.29%,硫酸消耗量降低了88.53%,而且废水中的COD和TOC分别降低了50.15%和64.39%。由此可见,综合利用—加压双相酸水解从新鲜黄姜中萃取薯蓣皂苷元的新方法是一种更为清洁和高效的途径,具有较好的发展潜力,在工业应用中应用前景良好。
[Abstract]:Diosgenin is a very important precursor compound in drug production. It is the main raw material for the synthesis of contraceptives, cortisone, sex hormone, protein assimilation hormone and so on. Therefore, it has the reputation of "medicinal gold" and "the mother of hormone" in the medical field. In addition, the pharmacological study shows that diosgenin has a variety of bioactivity. In our country, the diosgenin in the plant is mainly in the form of saponins, and the production of diosgenin must make the glycosides hydrolyze. The production of diosgenin must be hydrolyzed. The production of diosgenin in Huang Jianghe was produced by traditional methods. Diosgenin leads to serious environmental pollution, the non saponins can not be used, and the yield of diosgenin is low. The purpose of this study is to establish a more clean and efficient method to produce diosgenin from Zingiber rhizome and Dioscorea, and to improve the utilization efficiency of medicinal Dioscorea. The main research work has been completed. (1) the method of determining the content of diosgenin by HPLC-UV was established to establish the HPLC chromatographic analysis conditions, that is, the chromatographic column is Waters XBridge Shield RP18 (4.6mm x 150 mm, 5 u m); the mobile phase is acetonitrile: water (75:25); the flow rate is 1 mL/min; the detection wavelength is 203 nm; the sample volume is 20 mu; the column temperature is 15; the analysis time is 15 under this condition, Dioscorea The saponins could be separated from the interfering components (R1.5). After that, a methodological study was conducted, including linear, precision, stability, repeatability and recovery. The results showed that diosgenin had a good linear relationship in the range of 0.002~1.024 mg/mL (r2= 0.9999); the precision experiment RSD=1.02% (n=6); RSD=1.78% (n=6) for the repeatability experiment. The average recovery rate was 96.2% (RSD=1.17%), 100.4% (RSD=2.47%) and 101.6% (RSD=1.09%), and the test product solution was determined in 24 hrs. RSD=1.93%. can be seen from this, the established HPLC analysis method is stable and reliable, and can be used in quantitative analysis of diosgenin. (2) diosgenin extracted from rhizome of Dioscorea zingiberensis by direct pressure double phase acid hydrolysis First, the orthogonal experimental design was used to optimize the conditions of the pressure biphasic acid hydrolysis reaction. The effects of four factors, reaction temperature, sulfuric acid concentration, reaction time and liquid ratio, on the yield of diosgenin were investigated. The effects of 4 factors were evaluated by the difference analysis and variance analysis, and the optimum extraction conditions were obtained. After that, the results were verified. The optimum extraction conditions were compared with two traditional methods and pre fermentation biphasic acid hydrolysis. In addition, the Diosgenin was separated and purified by silica gel column chromatography and recrystallization, and the purity of diosgenin was detected. The results showed that the purity of diosgenin was obtained. The results showed that the purity of diosgenin was obtained. When the reaction temperature is 140oC, the concentration of sulfuric acid is 0.2mol/l, the reaction time is 1.5hrs, the yield of diosgenin is up to 2.5g/50ml, the yield of diosgenin is reached to 2.21%. by the new method, the yield of diosgenin is 63.7%, 70.4 and 20.8% higher than that of the two traditional methods and the prefermentation biphasic hydrolysis method, respectively, and the sulphuric acid concentration required is only about 1/10 of the traditional method. It can be seen that the yield of diosgenin extracted from Dioscorea Zingiberis by direct pressure biphasic acid hydrolysis is higher, the amount of sulfuric acid is reduced obviously and the reaction time is shortened. (3) the extraction of diosgenin from Dioscorea Zingiberis by the extraction of total saponins with pressure biphasic acid hydrolysis method first extract the total saponins from the Dioscorea from diosgenin by 70% ethanol solution and then the extraction of the total saponins in the Dioscorea The Diosgenin was hydrolyzed with pressure biphasic acid hydrolysis method to produce diosgenin. Four factors were investigated by single factor experiment, including the ratio of material to liquid, concentration of sulfuric acid, reaction temperature and reaction time, and the optimum reaction conditions were optimized by orthogonal design. The ratio of material to liquid was 8g/50ml, and the concentration of sulphuric acid was 4 mu. The reaction temperature was 150oC and the reaction time was 2.5hrs. compared with the traditional method, the yield of diosgenin was 1.67%, increased by 66.02%, and the consumption of sulphuric acid was 1.497ml/g, and 97.00%. was reduced. The Total Saponins Extracted and pressurized biphasic acid hydrolysis could be used to extract diosgenin more efficiently and cleanly from the Dioscorea It also indicates that pressure biphasic acid hydrolysis is an effective way to produce diosgenin from Dioscorea plants. (4) comprehensive utilization of pressure biphasic acid hydrolysis method from fresh ginger extract diosgenin from diosgenin by physical method to recover starch and cellulose from fresh yellow ginger by physical method, and then extract crude saponins by pressure biphasic acid hydrolysis extraction Diosgenin (diosgenin). The effects of the ratio of material to liquid, the concentration of sulfuric acid, the volume of the solvent, the reaction temperature, the reaction time, the speed and the boiling process of petroleum ether on the yield of diosgenin were investigated in turn, and the optimum reaction conditions were optimized. After that, the yield of diosgenin, the consumption of sulphuric acid and the production of the wastewater were produced. The evaluation of oxygen demand (COD) and total organic carbon content (TOC) is compared with the new method and direct pressurized biphasic acid hydrolysis and traditional method. The results show that the concentration of sulphuric acid is 2 mu l/ml, the extraction reagent is 30ml petroleum ether (90~120oc) and the reaction temperature is 130oc when the ratio of the feed to liquid is 1g/100ml (the mass of the crude saponins / sulphuric acid solution). When the reaction time is 2hrs and the stirring speed is 100r/min, the yield of diosgenin can be reached to 4.17%.. Compared with the traditional method, the yield of diosgenin is increased by 22.29%, the consumption of sulphuric acid is reduced by 88.53%, and the COD and TOC in the wastewater are reduced by 50.15% and 64.39%. respectively. The synthetic utilization of pressure diphasic acid hydrolysis from fresh yellow ginger The new method of extracting diosgenin is a more clean and efficient way. It has good potential for development and has good application prospects in industrial applications.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284.1

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