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Alternative TitleSeparation and purification of total saponin from seed kernel of Xanthoceras sorbifolia Bunge
Thesis Advisor王晓东
Degree Grantor中国科学院研究生院
Degree Discipline生物工程
Keyword文冠果   皂苷   三液相萃取   大孔树脂   分离纯化
Abstract选择了石油醚/乙醇/硫酸铵三液相体系作为分离文冠果油脂和皂苷的溶剂体系,考察了三液相体系中皂苷分配的影响因素,优选了三液相体系各成分含量组成,在超声辅助条件下优化了提取工艺。主要结论如下: 文冠果种仁粉经三液相萃取之后,油脂主要分配在上相石油醚中,种仁皂苷主要分配在富含乙醇的中相,其它极性强的杂质蛋白质,多糖等则主要集中在富盐的下相,残渣则在中相和下相之间,可通过过滤收集。 考察了三液相体系提取油脂和皂苷过程中的影响因素,皂苷分配系数K和回收率R均随硫酸铵盐浓度和乙醇浓度增加而增加,最适pH值为7.5、石油醚用量40%,温度对于相分配效果没有显著影响。从不同的体系中筛选出了最优三液相体系:40%石油醚/25%乙醇/22%硫酸铵。 响应面法优化了超声辅助三液相(Three-liquid-phase extraction,TLPE)提取皂苷的工艺条件:最优条件为24min,液固比25mL/g,温度为40℃。皂苷得率预测值为5.16mg/g,实际得率为(5.26±0.15)mg/g,与模型预测值相比相对误差在5%以内。 超声辅助三液相法得油率为80.1%,对所得油脂组分分析,该油脂品质较好,质量稳定,与传统法获得的油脂比较,不饱和脂肪酸的含量没有显著差异。 研究了大孔树脂分离纯化文冠果总皂苷的工艺方法。结果表明:在所选8种树脂中,ADS-7大孔树脂有较好的吸附效果。最优纯化工艺为:上样液浓度约为0.6mg/mL,上样液pH为4.5,吸附洗脱流速均为4BV/h;洗脱时,先用2BV的去离子水,然后用3BV的70%乙醇溶液洗脱,再用2BV的90%乙醇洗脱,解吸率达95.8%,皂苷含量达到32.2%,比纯化前高4.4倍,比正丁醇萃取高1.2倍。 体外活性实验表明,皂苷纯化物有清除亚硝酸盐的活性,其IC50为3mg/mL;纯化物有抑制肝癌细胞HUH-7活性,IC50为43.4μg/mL。
Other AbstractThree-phase system of the petroleum ether/ethanol/ammonium sulfate was chosen as an extraction and separation solvent system of oil and saponins. The influence factors of saponin partition were investigated in the three-liquid-phase extraction (TLPE) process. The extraction conditions in three-phase system were optimized under the assistance of ultrasonic. After seed powder of Xanthoceras sorbifolia was extracted by three-liquid-phase, oil was primarily distributed in the top phase with petroleum ether, saponins were mainly distributed in the middle phase with ethanol. Other strong polar impurities such as protein, polysaccharide etc. were mostly concentrated in the bottom phase with salt. Residue suspended in interphase was collected through filtering. The factors influenced the extractions of oil and saponins in the three-liquid-system were examined. The value of K and R increased with ammonium sulfate and ethanol concentration. The optimized pH value was 7.5. The dosage of petroleum ether was 40%(w/w). There was no significant effect of temperature on separation. In different systems, the optimal three-phase-system of 40% petroleum ether/25%ethanol/22% ammonium sulfate was selected as solvent of extraction experiments. The response surface method was used to optimize the TLPE parameters based upon the results of single factor experiments. The optimal conditions for extracting saponin were as follows: extraction time was 24min, the ratio of liquid to solid was 25:1, extraction temperature was 40℃. The predictive value of saponin yield was 5.16mg/g. The actual yield was (5.26±0.15)mg/g. The relative error was within 5% compared with the model prediction. Through the oil component analysis obtained from UA-TLPE system, the oil yield was 80.1% and the oil quality was good and stable. Compared the oil obtained from UA-TLPE with that from traditional method, no significant differences were found in the contents of unsaturated fatty acids. The separation and purification parameters of total saponin by macroporous resins were optimized. ADS-7 resin was chosen due to its excellent absorption and selectivity. The optimal purification conditions were confirmed as follows: sample concentration 0.6mg/mL at pH 4.5, and the volumetric flow rate of 4BV/h for elution and adsorption. The elution process was as follow: after 2BV of deionized water were used, 3BV of 70% ethanol and 2BV of 90% ethanol were used as eluents in turn, desorption flow rate was 4BV/h. Under these conditions, the desorption rate reached 95.8% and the purity of total saponin reached 32.2%, which was 4.4 times higher than that before purification and 1.2 times higher than that obtained by butyl alcohol extract.The activity experiment revealed that the purified saponins had a certain activity of scavenging the nitrite with IC50 3mg/mL and inhibiting growth of liver cancer cell with IC50 43.4μg/mL.
Document Type学位论文
Recommended Citation
GB/T 7714
王清亭. 文冠果种仁皂苷分离纯化研究[D]. 中国科学院研究生院,2013.
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