天山雪莲质量评价与有效成分的提取分离研究
文献类型：学位论文
| 作者 | 姚凌云 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-04-06 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 赵兵 |
| 关键词 | 雪莲 指纹图谱 超声提取 有效成分 多糖 |
| 其他题名 | The quality assessment of Saussurea involucrata and studies on the extraction and separation of its effective constituents |
| 学位专业 | 生物化工 |
| 中文摘要 | 天山雪莲(Saussurea involucrata)收载于《中华人民共和国药典》(2010年版一部)，是一种濒危珍稀药用植物，其药理作用有抗炎镇痛、抗衰老及抑制癌细胞增生等。科研工作者们一直将雪莲植物保护及活性成分的筛选视为研究热点，但对雪莲植物资源的质量评价及活性成分的分离制备研究较少；现存提取工艺产率低、能耗高、缺乏完善的提取过程动力学研究；随着野生雪莲资源的日渐萎缩，种植雪莲能否替代野生品种，对其资源质量评价、成分分析及生物活性等尚缺乏系统研究，这些问题都严重阻碍了雪莲植物资源的合理开发及可持续发展。本文以野生和种植的天山雪莲为对象，借助中药指纹图谱、超声提取、大孔树脂及高速逆流分离等技术，系统研究了雪莲植物资源的质量评价方法、生物活性成分的提取与分离工艺，旨在为雪莲资源的综合利用奠定理论基础。本文主要研究内容包括： (1) 雪莲质量评价体系的建立与指标性成分含量测定以天山雪莲为研究对象，收集了22个不同产地样品。采用高效液相色谱法对雪莲中的绿原酸、黄酮类等成分进行HPLC指纹图谱分析，获取了反映雪莲化学成分的色谱峰数据。选择推荐品雪莲建立HPLC指纹图谱的共有模式，计算样品与共有模式的相似度，根据相似度结果对雪莲的质量进行评价。收集不同生长期的种植雪莲苗，根据建立的指标性成分含量测定方法，评价雪莲种植过程中初生代谢产物的积累状况并指导雪莲的采收。 (2) 超声提取雪莲黄酮过程动力学与提取工艺优化以雪莲黄酮为目标成分，以Fick定律为基础建立植物有效成分超声提取动力学模型，从实验和模型角度对超声强化雪莲植物资源提取的传质动力学进行了研究。以绿原酸、紫丁香苷和芦丁3种雪莲中的代表性化合物的提取率为指标，采用响应面分析法对野生雪莲的超声强化乙醇提取工艺进行了系统的研究，并建立了提取效果预测模型。通过方差检验分析，证明了该模型具有较好的精确度、拟合度和响应性，通过实验验证预测值与实验值相差较小，可以用来对实验结果进行预测。考察了乙醇提取后的雪莲残渣超声提取多糖的工艺条件，采用响应面设计方法对多糖提取的过程进行了优化，并建立了多糖提取的预测模型。 (3)从种植雪莲中提取、纯化雪莲总黄酮的研究雪莲中黄酮类化合物的最佳提取工艺为：物料粉碎为60目，乙醇浓度为60%，提取温度25 °C，超声功率为900 W，液固比25.5:1，提取时间为33 min。验证实验提取率平均值为52.95 mg/g，与预测值基本吻合，说明该模型具有较好的可预测性。实验筛选了X-5大孔吸附树脂进行纯化，黄酮初始浓度为0.55 mg/mL上柱，依次用30%的乙醇2 BV (Bed volume)、80%的乙醇溶液5 BV以2 mL/min的流速进行洗脱，收集的洗脱液跟踪检测后合并。富集的黄酮含量达81.5%，黄酮回收率为83.6%。 (4) 高速逆流色谱法分离纯化雪莲中的活性化合物采用乙酸乙酯:正丁醇:水=3:2:5溶剂体系，从种植雪莲中分离得到3种化合物，分别为紫丁香苷(Syringin)、芦丁(Rutin)、高车前素(Hispidulin)，HPLC峰面积归一法计算其纯度分别为95.73%、97.56%、93.28%。在野生雪莲的逆流色谱分离中，采用正己烷:乙酸乙酯:甲醇:水=2:3:0.5:4、正己烷:乙酸乙酯:甲醇:水=1:6:1.5:7.5与正丁醇:乙酸乙酯:水=1.5:3.5:5等溶剂体系，分离得到6个化合物(异槲皮素、紫丁香苷、芦丁、槲皮素、高车前素和一未鉴定化合物)。 (5) 雪莲多糖的分离纯化与结构研究经超声水提、乙醇沉淀、脱蛋白、脱色、透析得到雪莲水溶性多糖粗品CPS和WPS，用DEAE-cellulose柱进行柱层析分离，依次用0~1.0 mol/L的NaCl溶液各200 mL洗脱，收集主要洗脱峰，得CPS1-2、CPS2-3和WPS2-2、WPS3-4、WPS4-2等5种纯化多糖。并对多糖的分子量、单糖组成及红外光谱特征等进行了研究。 (6) 雪莲成分抗氧化活性研究对野生雪莲和种植雪莲中的黄酮、多糖等10种不同成分进行了体外抗氧化活性测定。雪莲黄酮和雪莲多糖的体外抗氧化实验结果表明，对•OH、DPPH、•O2- 清除作用明显，且具有较好的还原力，且随着质量浓度的升高而升高。 |
| 英文摘要 | Saussurea involucrata is a kind of ethnic medicine listed in Pharmacopoeia of the People’s Republic of China (Edition 2010, Volume 1), its pharmacological effects are anti-inflammation, analgesia, anti-aging, contraception and so on. This endangered plant attracts much attention of pharmaceutical researchers in its bioactivities and cultivation. But the quality evaluation of this medicinal plant and separation of the bioactive ingredients were less investigated. The traditional extraction method is time-consuming and high energy consumption, and there is a lack of extraction kinetic studies. Otherwise, whether cultivated S. involucrata can replace the wild plant used in medicine, quality evaluation, composition analysis and biological activities need to be investigated for sustainable use of this medicinal plant. The HPLC fingerprints of S. involucrata, ultrasound-assisted extraction and separation technology were studied in this paper, to provide advanced technical support for comprehensive utilization of S. involucrata resources. The main contents are as follows: (1) Establishment of quality evaluation method and determination of active ingredients in S. involucrata HPLC fingerprint analysis of S. involucrata was established, 22 samples were collected and examined to obtain the medicinal chemistry data, including chlorogenic acid, flavonoids and other ingredients. Common pattern was obtained through the selected medicinal products in S. involucrata, and the quality of an unknown sample was assessed based on similarity evaluation criteria. Cultivated S. involucrata seedlings were collected at different growth stages. The target components were analyzed to assess the quality of the medicinal plants and guide for the herb harvesting. (2) Integrated ultrasonic-assisted extraction kinetics of flavonoids from S. involucrata Based on the Fick’s law, the extraction kinetic models of flavonoids from S. involucrata were established for the first time. An efficient UAE method for the extraction of bioactive compounds from S. involucrata was optimized by the response surface methodology (RSM). The extraction model was established using the ectraction rate of hlorogenic acid, syringin and rutin as response values. The model showed good accuracy and fitness through verification experiments. S. involucrata residue after extraction by ethanol was further extracted with water assisted by ultrasound for the extraction of polysaccharides. RSM design method was employed to optimize the extraction process, and an extraction model was established to evaluate the extraction rate of polysaccharide from S. involucrata. (3) Optimization of the extraction and purification of flavonoids from cultivated S. involucrata by macroporous resin The optimized extraction process was as follows: particle size was 60 mesh, ethanol concentration was 60%, extraction temperature was 25 °C, ultrasonic power was 900 W, liquid-solid ratio was 25.5:1 (mL/g), extraction time was 33 min. Nine different resins were used for separation of flavonoids from S. involucrata. The experimental results showed that a resin labeled X-5 offered higher adsorption and desorption capacities and higher adsorption speed for syringin and flavonoids than other resins, and its adsorption data fitted to Freundlich isotherm. The dynamic adsorption and desorption experiments were carried out in the column packed with X-5 resin to optimize the separation process. Enriched flavonoids extract without syringin, and an enriched syringin extract with 80.3% purity could be separated from crude S. involucrata extracts in one run. The content of enrichment flavonoids was 81.5%, flavonoid recovery was 83.6%. (4) High-speed counter current chromatography (HSCCC) separation and purification of active compounds from S. involucrata The crude extract of cultivated S. involucrata was separated and purified by HSCCC using ethyl acetate–n-butanol–water (3:2:5, v/v/v) solvent system. Under optimal HSCCC conditions, syringin, rutin and hispidulin were obtained by one-step HSCCC separation, with purities of 95.1%, 98.7%, and 96.2%, respectively. Six compounds (isoquercetin, rutin, syringin, quercetin, hispudlin and an unkow compound) were isolated from wild S. involucrata, hexane: ethyl acetate: methanol: water = 2:3:0.5:4, hexane: ethyl acetate: methanol: water = 1:6:1.5:7.5 and n-butanol: ethyl acetate: water = 1.5:3.5:5 solvent systems were used for HSCCC. (5) Purification and composition of the polysaccharide from S. involucrata Polysaccharides from cultivated S. involucrata (CPS) and wild S. involucrata (WPS) were purified. Five major fractions (CPS1-2, CPS2-3, WPS2-2, WPS3-4 and WPS4-2) were examined for their molecular weights, monosaccharides compositions. And IR spectra of purified fractions were also investigated. (6) Antioxidant activity of S. involucrata Different scavenging activities on superoxide radical, DPPH radical, hydroxyl radical and reducing power were observed in nine different fractions (polysaccharide and flavonoids) from S. involucrata at tested concentrations. And the scavenging rate and reducing power increased with the increase of the concentration. |
| 语种 | 中文 |
| 公开日期 | 2013-09-25 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1874]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 姚凌云. 天山雪莲质量评价与有效成分的提取分离研究[D]. 中国科学院研究生院. 2012. |
入库方式： OAI收割
来源：过程工程研究所
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