New Extraction Technologies and Separation with High-speed Countercurrent Chromatography of the Active Constituents from Licorice

中药有效成分研究是中药物质基础研究的主要内容之一，如何将已知或未知的有效成分高效率地提取出来则是中药有效成分研究的关键所在，也是中药开发和中药现代化研究的关键技术之一。目前中药物质基础研究中存在的最大问题是缺乏客观、定量的品质标准和用药依据，因此，提取、分离、纯化中药的主要活性成分，是现阶段中药基础性研究的核心。基于此，本论文工作以中药甘草为研究对象，探索了更有效的中药有效成分的提取、分离和纯化方法，发展了甘草有效成分的微波辅助提取和多级逆流提取两种新型的提取技术以及高速逆流色谱（HSCCC）分离纯化方法。其意义在于为中药甘草的物质基础研究和甘草资源的深度开发提供有价值的参考依据和方法，为中药...Effective and efficient analytical technology for the extraction and isolation of active components in herbal plants or extracts is an important area of research in the current push towards the modernization of TCM (Traditional Chinese Medicine). In this thesis, several novel extraction techniques including microwave-assisted extraction (MAE), multistage countercurrent extraction (MCE), and high-s...学位：理学博士院系专业：化学化工学院化学系_分析化学学号：B20012500

Rapid Quality Control of Licorice in Its GAP Research

目的　建立甘草 GAP过程中的快速质量检验方法 ,为甘草药材的快速质量检测提供方法依据。方法　提出甘草中水溶性有效物质群的概念并建立其含量的比色测定方法。以甘草酸作为定量的标准物质 ,采用香草醛 -浓硫酸试剂作显色剂 ,用便携式小型光谱仪于 5 6 0 nm处检测。结果　回归方程为 Y=1.174 7C+0 .0 873,相关系数 r=0 .970 5 (n=3) ,回收率为 92 %～ 99% ,在 0 .0～ 0 .96 mg/m L 范围内仪器读数与甘草酸浓度呈线性关系。结论　该方法简单、快速、廉价 ,能够准确地反映甘草质量的优劣 ,适合在准确度要求相对较低的甘草生产基地推广使用Objective To develop a rapid method for the quality control of licorice in its GAP research.Methods Water-soluble triterpenoid saponins were defined as the active fraction in licorice and a colorimetric method using LED-based portable device has been developed for the first time to measure the content of this active fraction. The method was based on the specific color reaction occurring between triterpenoid saponins and vanillin-sulfuric acid reagent. Visible light absorption of 560 nm was chosen as the detection wavelength.Results The operating calibration curve(Y=1.174 7C +0.087 3 mg/mL) was found to be linear at the range of 0.0～0.96 mg/mL glycyrrhizic acid in water (r=0.970 5). The recovery is 92～99%.Conclusion The method is simple, rapid and low-cost and is recommendable to evaluate the quality of Glycyrrhiza uralensis Fisch. in its GAP research.内蒙梁外甘草 GAP研究与开发;; 国家自然科学基金 2 0 0 3重点项目 ( 2 0 2 3 5 0 2 0

Application of pattern recognition technology in classification of Radix et Rhizoma Glycyrrhizae of different sources

目的:建立不同来源甘草药材的模式识别分类方法。方法:通过 HPLC 快速获得19个不同来源甘草药材样品的色谱指纹图谱,色谱条件固定相为 Kromasil KR100-5 C_(18)(150mm×4.6mm),流动相为乙腈-0.05%三氟乙酸溶液,梯度洗脱为乙腈20%(0min)→40%(5min)→50%(10min),流速为0.8mL·min~(-1),检测波长为254nm,从每个色谱指纹罔中提取出325个特征变量,采用聚类分析对甘草药材样品进行相似性评价,并应用 PLS 方法建立起不同植物种、不同种植方式甘草的2个分类回归模型。结果:采用分类模型对不同植物种、不同种植方式甘草进行判别,2个分类回归模型的分类准确率均为100%。结论:此方法可用于不同来源甘草药材的分类研究,本文以 HPLC 指纹图谱结合化学计量学知识,为不同中药样品的质量评价提供了一种分类方法。Objective:To establish a method for pattern recognition classification on Radix et Rhizoma Glycyrrhizae of different sources.Methods:HPLC system was applied to obtain the chromatograms of the 19 Radix et Rhizoma Glycyrrhizae samples.The Kromasil KR100-5 C_(18)(150 mm×4.6 mm)column was used.A mixture of acetonitrile 0.05% trifluoroacetic acid solution was used as the mobile phase with a flow rate of 0.8 mL·min~(-1) and the gradi- ent mode was acetonitrile 20%(0 rain)→40%(5 min)→50%(10 min).The detection wavelength was 254 nm. The 325 characteristic variables were obtained from each chromatogram.The clustering analysis was applied to eval- uate the similarity of the chromatograms.Then partial least-squares regression method was applied to build the pre- diction models of classification which were used to distinguish Radix et Rhizoma Glycyrrhizae of different species and cultivation modes.Results:The classification accuracy of the two prediction models of classification was 100% for Radix et Rhizoma Glycyrrhizae of different sources.Conclusion:This method is practicable in the pattern recog- nition classification on Radix et Rhizoma Glycyrrhizae of different sources.The developed technique could be further applied to the classification of different traditional Chinese medicine(TCM).福建省自然科学基金(C021006);; 福建 GAP 关键技术研究(2002Y024

Microwave extraction of glycyrrhizic acid from Glycyrrhiza uralensis

目的　研究利用微波萃取技术提取甘草中甘草酸的方法。方法　采用正交试验考察提取温度、提取时间、微波功率对甘草酸含量和提取总时间的影响 ,确定微波萃取甘草中甘草酸的最佳工艺条件。在优选出的微波萃取最佳工艺条件下 ,考察了提取溶剂对甘草酸含量的影响 ,并与超声波提取法、室温冷浸法和索氏提取法比较。结果微波提取的最佳条件为以 0 5 %氨水为提取溶媒 ,微波功率为 2 0 0 0W ,体系温度升至 6 0℃后保温提取 4 0min。微波萃取 5 4min与索氏提取 4h、室温冷浸 4 4 3h的甘草酸得率相当。结论　微波萃取具有快速、高效、节能、选择性好的特点 ,可用于中草药有效成分的提取 ,值得推广应用。Object A novel microwave-heated extraction (MHE) method was studied for the extraction of glycyrrhizic acid from Glycyrrhiza uralensis Fisch. Methods Several factors, such as temperature, time and microwave power were investigated and the appropriate MHE conditions were obtained from the orthogonal test. Under the optimum conditions, the optimal solvent was selected and the MHE was compared with ultrasonic extraction, leaching at room temperature and Soxhlet extraction,. Results The optimum conditions of MHE is extracting for another 40 min in 0.5% ammonia water after heated to 60 ℃ by microwave of 2 000 W. Yield of glycyrrhizic acid was about equal to that of Soxhlet extraction for 4 h, and that of leaching at room temperature for 44.3 h. Conclusion The MHE method is fast, efficient, energy-saving and high-selective, which is recommendable to the application to active compounds extraction from Chinese herbal medicines.福建省中药质量标准重点项目 (2 0 0 0F0 0 1

Rapid Determination of Triterpenoid Alcohol Ketones in Alisma orientalis

建立了利用便携式小型光谱仪测定中药泽泻中三萜醇酮类化合物总量的比色测定方法.本方法采用具有C 3羰基的甾酮类化合物的专属性Zimmerman显色反应,检测波长为560nm.以23 乙酰泽泻醇B的吸光度和浓度作工作曲线,回归方程为A=2.1009C+0.2116(mg/mL),相关系数为0.9997,23 乙酰泽泻醇B的浓度在0～160mg/mL范围内符合比耳定律,表观摩尔吸收系数ε=5.4×102L·mol-1·cm-1,检出限为11.143mg/mL,回收率为93.0%～109%.该方法简单、快速、廉价,已成功应用于福建地道药材泽泻GAP过程质量控制及泽泻药材品质的评价,指导泽泻药材的生产,具有重大的经济效益和社会意义.The use of LEDbased portable device for the colorimetric determination of triterpenoid alcohol ketones in Alisma orientalis has been established.The method is based on Zimmerman reaction, which is the specific reaction occurring in 3carbonyl of sterones. 560 nm was chosen as the detection wavelength. The operating curve (A=2.100 9 C+0.211 6 mg/mL)) was found to be linear over the range of 0～160 μg/mL acetate alisol B in ethanol (r =0.999 7). The molar absorptivity is 5.4×102 L·mol-1·cm-1.The detection limit is 11.143 μg/mL and the recovery is 93.0%～109%. The simple, rapid and lowcost method has been successfully applied to evaluate the quality of Alisma orientalis in our GAP (Good Agricultural Practice) research and practice in Fujian province.福建省中药标准化项目(2000F001);; 福建省泽泻GAP重大项目及重点项

Separation of ethanol extract of Rhizoma of Alisma Orientalis by High Performance Capillary Electrophoresis (HPCE)

采用高效毛细管电泳的胶束电动毛细管色谱的分离模式(MEKC),对泽泻的乙醇(95%)提取物进行分离研究。以十二烷基硫酸钠(SDS)胶束为准固定相,硼砂为缓冲体系,甲醇为有机添加剂,考察了SDS浓度、硼砂浓度、pH、甲醇用量、电泳电压、电泳温度等对分离的影响。结果表明:硼砂缓冲体系为运行缓冲溶液(pH:9.18～9.20)、硼砂浓度30mmol/L、SDS浓度45mmol/L、甲醇体积分数30%,分离电压25kV、分离温度25℃时分离效果最好。在此条件下,得到分离度和重现性均较好的泽泻乙醇提取物的HPCE色谱图。The separation of the compounds extracted from rhizoma of Alisma orientalis (Sam.) Juzep using 95% ethanol by micellar electrokinetic capillary chromatography (MKCC) with sodium dodecyl sulphate (SDS) as the micellar phase, borax as a buffer and methanol as an organic additive was studied, which established a fingerprint technique for the rhizoma of Alisma orientalis. The effects of SDS micellar and borax concentration, pH buffer, methanol volume fraction, applied voltage and temperature on the separation were investigated by the orthogonal test using chromatography optimization function (COF) as the evaluation index. It was found that the optimal separation conditions of these components might be achieved by using a fused silica capillary with 60 cm long and 75 μm ID, in which 30 mmol/L borax buffer solution (pH 9.20) containing 45 mmol/L SDS and 30% methanol was filled under the voltage of 25 kV and a temperature of 25 ℃. HPCE fingerprint of the rhizome of Alisma orientalis with perfect separating size and repeatability could be expected. Thus, the method provides a basic information for the whole quantity control of the rhizoma of Alisma orientalis in the products

Study on Optimal Harvest Time of Semi-wild Glycyrrhiza uralensis at Liangwai,Inner Mongolia

目的 :研究甘草中甘草酸的积累动态 ,确定内蒙古梁外地区育苗移栽的半野生甘草最佳采收期。方法 :用HPLC法测定不同时间采集的不同株龄的半野生甘草中甘草酸含量。结果 :甘草中甘草酸含量 6～ 7月份最高 ,8月份开始逐渐降低 ,11～ 12月最低 ,翌年 3～ 4月份开始逐渐增加 ,直到 6～ 7月份达最高峰 ;甘草在移栽后的前三年 ,甘草酸含量无明显增加 ,第四年的各个月份的甘草酸含量均远大于头三年。结论 :育苗移栽的半野生甘草的最佳采收期为移栽后的第四年 7～ 8月份Objective: To study the dynamic accumulation of glycyrrhizic acid(GA) in semi wild Glycyrrhiza uralensis Fisch. so as to determine its optimal harvest time Methods: The content of GA in the plant samples was determined by HPLC Results: In the same growth period, the content of GA in the plant reached its highest peak in June or July, decreased gradually from August, and reached the lowest in November or December. Then the content of GA increased gradually from next March, and finally reached the highest in June or July again There was no obvious difference between the content of GA in the one year old and the two year old Glycyrrhiza uralensis But the content of GA in the three year old increased markedly, and its GA content was much higher than that of the one or two year old Conclusion:The optimal harvest time of semi wild Glycyrrhiza uralensis is in July or August after transplanted three years国家自然科学基金 2 0 0 3年重点项目( 2 0 2 3 5 0 2 0

泽泻药材的HPCE指纹图谱研究

采用高效毛细管电泳的胶束电动毛细管色谱的分离模式(MEKC),对泽泻的乙醇(95%)提取物进行分离研究。以十二烷基硫酸钠(SDS)胶束为准固定相,硼砂为缓冲体系,甲醇为有机添加剂,考察了SDS浓度、硼砂浓度、pH值、甲醇的用量、电泳电压、电泳温度等对泽泻的分离效果。结果表明:硼砂缓冲体系为运行缓冲溶液的pH值9.18～9.20、硼砂浓度为30mol/L、SDS浓度45mmol/L、甲醇30%,分

Separation of ethanol extract of Rhizoma of Alisma Orientalis by High Performance Capillary Electrophoresis (HPCE)

采用高效毛细管电泳的胶束电动毛细管色谱的分离模式(MEKC),对泽泻的乙醇(95%)提取物进行分离研究。以十二烷基硫酸钠(SDS)胶束为准固定相,硼砂为缓冲体系,甲醇为有机添加剂,考察了SDS浓度、硼砂浓度、pH、甲醇用量、电泳电压、电泳温度等对分离的影响。结果表明:硼砂缓冲体系为运行缓冲溶液(pH:9.18～9.20)、硼砂浓度30mmol/L、SDS浓度45mmol/L、甲醇体积分数30%,分离电压25kV、分离温度25℃时分离效果最好。在此条件下,得到分离度和重现性均较好的泽泻乙醇提取物的HPCE色谱图