摘要
生物碱作为我国传统中药苦参(Sophora flavescens Ait.)的主要有效成份,具有广泛而显著的药理活性,特别是其抗癌作用备受关注,而因业内企业受当前技术水平所限,使得这类药物不断增长的社会需求难以得到满足。本文在综合分析了已有的各种生物碱的提取分离方法的基础上,确定了一套以低成本,高效率为目标的提取分离过程技术研究方案,并从提取、总生物碱分离到主要单体生物碱的分离纯化进行了详细的实验研究,主要研究内容如下:
1、提取分离以分析检测为先行,故本文首先探讨了适宜本物系进行定性定量分析生物碱含量的薄层色谱分析条件,选用了相对环境友好型清洁试剂代替了传统的毒性较高的苯C6H6和甲醇CH3OH等混合展开剂,并在前人的研究基础上以二次回归正交旋转组合设计优化了展开剂配比,选定预分离的4种主要生物碱中差别最小的槐果碱SC和苦参碱MAT的Rf值差值为目标函数,采用统计软件SAS9.0进行统计分析,从而确定能够得到最佳分离效果的展开剂配比为:乙酸乙酯/无水乙醇/氨水=5.2/0.7/0.7。通过验证实验得出优化后的Rf值差值为0.079。如此优化建立起来的薄层色谱检测体系简捷、精确、稳定、对环境相对友好,并且可以作为柱色谱的先导,能够预试摸索出柱色谱的洗脱条件。
2、在生物碱的浸提过程中,以同样的统计学方法优化了苦参中生物碱的提取工艺:以苦参生物碱的提取率为评价指标,利用薄层色谱检测生物碱的含量,选定原料粒度、浸取液pH值、料液比、渗漉流速这四个主要影响因素为考察对象,在单因素实验的基础上采用四因素五水平的二次回归正交旋转组合设计,并以SAS9.0软件进行数据分析,建立了苦参生物碱的提取率与各影响因子的回归方程,得出最优的提取条件为:以pH1.81的稀HCl水溶液浸润68-84目的苦参12h后,以3.37mL·min-1的流速进行渗漉,收集5.35倍料液比的渗漉液。在此条件下总碱提取率为94%。
3、在生物碱总碱的分离过程中,以可见分光光度法检测了等电点法除蛋白质的效果,并考察了不同浓度的醋酸铅应用于去除苦参渗漉液中的黄酮、蛋白质、氨基酸、有机酸、鞣质等杂质成分的沉淀效果,而后选用萃取法使生物碱总碱成分得以进一步分离。结果表明:1000g苦参经4000ml pH2的稀HCl浸提后,应用等电点法去除蛋白质成分后调节pH7,以浓度为0.050mg·ml-1的醋酸铅沉淀杂质成分,离心后调节溶液pH8.7以去除残余的铅离子,再经CHCl3萃取后最终得到了透明无色的生物碱总碱溶液,其中含SC 0.2242g,MAT 0.4818g,SRI 0.1242g,OMT 2.4995g。
4、在生物碱单体的分离过程中,借用薄层色谱的分析条件,以硅胶柱层析法进行单体生物碱的分离,最终得到了纯度达99.99%以上的4种主要生物碱SC 223.5mg,MAT 479.9mg,SRI 123.8mg和OMT 2483.7mg。
最后,为拓宽其中最具代表性的生物碱MAT的应用领域,探索了MAT对于体外培养人前列腺癌细胞PC-3M的影响,以期为前列腺癌的治疗及开发新药提供参考:
以MTT法检测了不同浓度0.3g·L-1、0.5g·L-1、0.8g·L-1、1.0g·L-1、1.5g·L-1、2.0g·L-1、2.5g·L-1、3.0g·L-1、3.5g·L-1的MAT对PC-3M作用不同时间24h,48h,72h,96h的细胞抑制率。结果显示,MAT对PC-3M的抑制作用呈量—效关系和时—效关系,随着作用时间的延长和药物浓度的增加,PC-3M的存活率逐步降低。
As a main active constituent of traditional Chinese medicine herb, alkaloids possess many kinds of biological activities and could cure cancers. But applied circumscription of medicine is confined by technology, which makes this kind of medicine can not meet the increasing need of society. This article designed a low cost and high efficient technology in extraction and separation based on the comprehensive analysis of extant alkaloids’extraction and separation. Then we carried on detailed research and experiment from the extraction, separation of the main alkaloids to the separation and purification of the main individual alkaloid. The main research contents are as follow:
1. Detection is essential for abstraction and extraction. Hence condition of thin-layer chromatography in test must be optimizated first. In the detection, less toxic reagent was selected to make instead of more toxic one and the match of developers was optimized according to quadratic regression rotational combinatorial design with SAS9.0. The results showed that the optimal chromatographic condition is EAc/C2H5OH/NH3·H2O=5.2/0.7/0.7. Condition of thin-layer chromatography optimizated is simple, exact and less toxic. It can provide some reference for column chromatography.
2. Effects of crush size, acidity of extracts, percolate speed, and solid-solvent ratio on extraction yields of alkaloids in Sophora flavescens Ait. And they were detected separately. Based on the above results, a quadratic regression rotational combinatorial design was applied to establish a regression model and to estimate the optimal conditions for maximal extraction yield. The results showed that the optimal extraction conditions were infiltrated Sophora flavescens Ait. of 68-84 meshs 12h with pH 1.81 Hydrochloric Acid, then percolated with the speed of 3.37mL·min-1 and collected 5.35 times percolate liquid. Based on the best extraction conditions, the extraction rate reached 94% through the verification experiment.
3. Solution was adjusted to the isoelectric point to remove proteins, and sediment effects of different density of plumbi acetas to remove dopant, which includes anthoxanthin, protein, amino acids, polysaccharides, organic acid and tannin, were investigated. At last, we used CHCl3 to separate alkaloids from other components. After efference, solution was adjusted below pH 8.7 to make the remnant plumbum ion removed, meanwhile could educe alkaloids, and benefit its transfer from lye to CHCl3.
4. Alkaloids were segregated into monomeric component with gel silica column chromatography. At last, we abtained 4 kinds, which are SC 223.5mg, MAT 479.9mg, SRI 123.8mg and OMT 2483.7mg respectively.
Eventually, the effect of Matrine on human carcinoma of prostate cell strain PC-3M in vitro has been detected, which meets the demand of clinical medication and could provide some references for MAT served as a new medicine.
In MTT test, PC-3M were treated with 0.3g·L-1, 0.5g·L-1, 0.8g·L-1, 1.0g·L-1, 1.5g·L-1, 2.0g·L-1, 2.5g·L-1, 3.0g·L-1, 3.5g·L-1 MAT, and cultured for 24h, 48h, 72h and 96h respectively. The result showed a linear relationship of effect and affeced time and a linear relationship of effect and concentration. Survival rate gradually decreased with prolonged affeced time and increased concentrations.
引文
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