【作者】 方俊；

【导师】 王建华；

【作者基本信息】 重庆大学 ， 生物医学工程， 2009， 硕士

【摘要】 葛根是一种有丰富营养而且药理作用独特的药食两用资源,用途极为广泛。葛根中的异黄酮类物质作为葛根的活性成分,其药理保健功能和临床治疗作用正逐渐被人们所认识和关注。鉴于国内和国际市场迫切需要异黄酮,而目前对异黄酮的提取方法和纯化需要改进,本论文对葛根中主要活性有效成分异黄酮的提取、分离与包合作了系统的研究。实验研究内容和结果如下:1.采用L9(34)正交设计法研究葛根总黄酮乙醇提取工艺:乙醇浓度、提取时间、提取温度、固液比对葛根异黄酮提取率的影响,方差分析表明各因素显著性顺序为乙醇浓度﹥提取温度﹥固液比﹥提取时间,并根据正交计算结果确定葛根乙醇提取的最佳工艺为:60%的乙醇15倍量,70℃回流提取1小时。总黄酮提取率为93%。2.在对超声波辅助提取工艺的研究中,考察了不同超声频率对提取率的影响,结果表明:超声频率越高,总黄酮提取率就越大;且在超声频率为32kHz,其它条件不变的情况下,提取半个小时就可达到95%的提取率,效率明显高于乙醇回流提取。在对超声波提取葛根总黄酮的动力学研究中,分别对曲线进行方程模拟,结果表明Cubic曲线在一定程度上反映了超声波提取葛根总黄酮的动力学过程。3.采用静态与动态的吸附-解吸两种方法,利用紫外可见分光光度计测量葛根总黄酮的含量,研究不同的ADS系列大孔吸附树脂对总黄酮分离纯化的影响,结果表明ADS-8分离效果最好,其最佳工艺为上柱液浓度约为6.8 mg/ml,pH值5～6,吸附速率4 BV/h,400 ml蒸馏水洗去杂质,洗脱剂为70 %乙醇,洗脱流速2 BV/h,用量60 ml,经ADS-8处理的葛根总黄酮洗脱率可达94%以上。4.采用AKTA Prime层析系统分离葛根异黄酮,薄层层析法检测收集液,并用HPLC鉴定,结果表明该系统能有效地将葛根素与杂质分开,分离后的样品葛根素含量约为70%;由样品图谱与葛根素标准品图谱比较可以看出,样品中的葛根素未受到干扰,分离效果较好。5.采用连续递变浓度法确定葛根异黄酮PU与β-CD、HP-β-CD、M-β-CD、SPE7-β-CD的最佳包合摩尔比均为1:1,并用超声波法制备了各包合物;相溶解法研究了四种环糊精溶液对PU的增溶作用,并测得各条件下的平衡常数,研究表明,在25℃、37℃和45℃三种温度下,PU的浓度均随环糊精浓度的增加而呈线性增加,相溶解度曲线为AL型,验证了包合比为1:1,在实验范围内约增溶1～3倍,25℃的平衡常数较其它温度高,37℃时最低;在包合物的稳定性研究中,对各包合物和物理混合物的引湿性进行考察,结果表明,包合物的湿稳定性明显高于混合物。更多还原

【Abstract】 Pueraria is an important constituent for drug and food since it has a lot of nutriments and particular meditative effects and has been widely used. As the main active component of Pueraria,Puerarin has been paid more and more attention and known for its meditative health protection function and clinic cure effect. Flavnonoids are eagerly needed in either domestic or the international markets. The extraction and purification techniques are needed to improve at present. This paper systematical studied on the process of extraction,purification and clathration of the main component- the isoflavones. The content and result of this study as follows:1. L9(34) Orthogonal design was adopted to research the factors: concentration of ethanol,extraction time,extraction temperature and ratio of ethanol to materials. The results of variance analysis showed that concentration of ethanol﹥extraction temperature﹥ratio of ethanol to materials﹥extraction time,and according to the calculation results of the orthogonal design, it indicated that the best extracting condition was: 60% ethanol, ratio of ethanol to materials was 15, extract 1 hour under the temperature of 70℃. With the best extracting condition, the extraction rate of total flavonoids was 93%.2. Effect of ultrasonic frequency on extraction rate was studied in ultrasonic extraction research, the results indicate:the higher the ultrasonic frequency was, the greater the extraction rate of total flavonoids was. Under the 32kHz ultrasonic frequency and other conditions remain the same as reflux extraction with ethanol, it could reach 95% of the extraction rate after half an hour, significantly higher than the reflux extraction. In the kinetic research of the ultrasonic extraction, the equation of the curve simulation results show that the Cubic curve reflected the dynamic processes of the ultrasonic extraction on total flavonoids.3. The static and dynamic adsorption/desorption methods were used to research the effect of different ADS series macroporous resin, and the separation and purification process was evaluated by measuring the concentration of total flavonoid in the fractions with UV spectrophotometer. Results indicated the ADS-8 macroporous resin was the most effective compared with other macroporous resins. The optimum conditions were screened, which were 6.8 mg/ml corresponding to crude drug for concentration of extract, pH 5–6 with the adsorption speed 4BV/h; water volume for removing impurities 400 ml; and the concentration of the eluant ethanol 70% (V/V), volume of ethanol 60 ml with desorption speed 2 BV/h. By this method, the final contents of total flavonoids exceeded 94%.4. The AKTA Prime system was used to separate flavnonoids with the methods of TLC and HPLC. Results indicated that the system could separate puerarin from the impurity, the content of puerarin was about 70% after sepatating. Puerarin in the sample was not interfered from the chromatogram of example compared to that of pueraria standard.5. Method of sequential change of molar concentration has been uesd to ensure the molar ratio of the inclusion complex (PU andβ-CD、HP-β-CD、M-β-CD、SPE7-β-CD) were all 1:1, and then the four kinds of inclusion complex were prepared by using ultrasonic method. The phase solubility method was used to prove the solubility-improvement and the stability constants were determined under the temperature of 25℃, 37℃and 45℃. The results indicated: the solubility of PU increased linearly with the increase of CD concentration, which showed typical AL type phase solubility profile, it also proved the molar ratio was 1:1; the solubility of PU was 1～3 times more in a series of concentrations of cyclodextrin; the stability constants of 25℃is higher than any others, and that of 37℃is the lowest. In addition, the results of stability investigation indicated that the inclusion complex was more stable than the physical mixture.更多还原