中药肉苁蓉多糖和总苷的提取分离及质量控制方法研究

摘要肉苁蓉为多年生草本植物，分布于新疆、内蒙古、陕西、甘肃、宁夏等地，具有补肾阳、益精血、润肠通便的功能。

本论文研究内容主要包括以下4个方面:1、肉苁蓉多糖的研究肉苁蓉多糖是肉苁蓉的有效成分之一，本文采用水提醇沉法对多糖进行提取分离，通过紫外分光光度法测定粗多糖和可溶性多糖的含量。

结果表明，粗多糖和可溶性多糖的含量分别为4.56%和17.41%；在肉苁蓉多糖提取研究的基础上对肉苁蓉多糖部位进行了研究，通过调节醇沉阶段的含醇量进行分段，用蒽酮-硫酸法对各部分多糖部位进行了含量测定。

结果显示，用20%乙醇沉淀所得多糖的含量最高，达到样品量的9.71%，占总多糖的44.0%。

2、肉苁蓉中毛蕊花糖苷的研究采用超声辅助提取法对肉苁蓉有效成分进行提取，用高效毛细管电泳法对肉苁蓉中毛蕊花糖苷进行含量测定。

详细考察了分离电压在8～25 kV范围和硼酸-硼砂缓冲溶液的pH在8.0～10.0之间对毛蕊花糖苷的分离影响，得到最佳分离条件为：电压8kV，运行缓冲溶液为30mmol/L的硼酸-硼砂（pH=9.0），室温，检测波长为334nm。

毛蕊花糖苷的浓度在0.015～0.375mg/mL 范围内呈良好的线性关系，其线性回归方程y=13946x+560.32,(R2=0.9983) ，样品回收率为99.26%，RSD=1.16%，精密度RSD=0.96%。

通过上述研究，建立了毛蕊花苷的高效毛细管电泳含量测定方法，并用此方法测得肉苁蓉中毛蕊花糖苷的含量为样品的1.02～1.04%。

3、肉苁蓉中红景天苷的测定方法研究采用HPLC和HPCE两种方法对肉苁蓉中红景天苷的测定方法进行了研究。

用HPLC法对肉苁蓉中红景天苷的测定研究，采用色谱柱为十八烷基硅烷键合硅胶柱，流动相为乙腈∶水(1∶9)，流速为1.0mL/min，室温，红景天苷的检测波长为278nm。

红景天苷在0.055～0.44µg范围呈良好的线性关系，其线性回归方程y=91207x－1097, (R2=0.9953) ，样品的回收率为94.8%，RSD= 1.50%，精密度RSD=0.39%。

通过研究得出HPCE法测定红景天苷的最佳条件：分离电压8kV，运行缓冲溶液30mmol/L的硼酸-硼砂（pH=9.0），室温，检测波长为278nm。

红景天苷的浓度在0.007～0.14mg/mL范围呈良好的线性关系，其线性回归方程y=42915x－92.541,(R 2=0.9903) 。

样品的回收率为100.1%，RSD= 1.45%，精密度RSD=0.96%。

对两种分离测定红景天苷的方法进行了比较,结果显示高效液相色谱法与毛细管电泳法对肉苁蓉中红景天苷的分离效果均较好，测定结果无明显差异，其含量均为0.103～0.120%。

4、肉苁蓉的指纹图谱研究本文采用HPCE方法，对肉苁蓉样品的提取成分进行了分离分析，以肉苁蓉的主要有效成份毛蕊花糖苷作为评价指标，建立了肉苁蓉药材的HPCE指纹图谱，其分离度和重现性均较好，各峰相对峰面积和相对保留时间的RSD均小于5%，为肉苁蓉药材的质量标准的建立提供了科学依据。

关键词:肉苁蓉；多糖；毛蕊花糖苷；红景天苷；指纹图谱AbstractHerba Cistanche, as perennial herbs, distribute in Xinjiang, Inner Mongolia, Shaanxi, Gansu, Ningxia and other places, with kidney-yang, essence and blood, and Runchang purge function.This thesis includes four parts:1. Study on the polysaccharides of Herba Cistanch eCistanche deserticola polysaccharides are the active ingredients in Herba Cistanche. Polysaccharides were separated by using aqueous alcohol extraction method and the content of crude polysaccharide and soluble polysaccharide was determined with ultraviolet spectrometry. The results showed that the content of crude polysaccharide and soluble polysaccharide in Herba Cistanch e is 4.56% and 17.41% separately. Based on the study of the extraction of polysaccharide in Cistanche, polysaccharides content is the highest when 20% ethanol was used, which can go to up to 9.71% of sample and 44.0% of total polysaccharides.2. Study on the Acteoside of Herba Cistanch eUltrasound-assisted extraction of active ingredients was employed, and acteoside in Herba Cistanch e was determined by high performance capillary electrophoresis. After detailed study of the impact of the separation voltage in the range of 8 ~ 25 kV and boric acid - borax buffer solution of pH 8.0 ~ 10.0 on the separation of acteoside, the best separation conditions were as follows: voltage at 8kV, running buffer solution with 30mmol/L of boric acid-borax (pH = 9.0), room temperature, and detection wavelength at 334nm. Acteoside concentration in the range of 0.015 ~ 0.35mg/mL showed good linear relationship. The linear regression equation is y=13946x+560.32 (R2=0.9983)，and sample recovery yield was 99.26% with RSD(1.16%) andprecision of RSD(0.96%). Through this research, we established the measurement of acteoside by high performance capillary electrophoresis method, and the content of Mullein glycoside in cistanche was determined to be 1.02 ~ 1.04% by the above method.3. Study on the determination of salidroside in Herba Cistanch eThe use of HPLC and HPCE methods for the determination of salidroside in Herba Cistanch e was studied. For HPLC method, column loaded with octadecyl silane bonded silica, mobile phase of acetonitrile: water (1:9), flow rate 1.0mL/min at room temperature, and the detection wavelength at 278nm were employed. The results indicated that salidroside in the range of 0.055 ~ 0.44μg was in good linear relationship with the linear regression equation y=91207x－1097, (R 2=0.9953), and sample recovery yield was 94.8%, RSD = 1.50%, and precision degree of RSD = 0.39%.Through our study, the best conditions for HPCE method were as follows: separation voltage at 8kV, running buffer 30mmol/L of boric acid-borax (pH = 9.0), room temperature, and detection wavelength at 278nm. The results showed salidroside concentration in the range of 0.007 ~ 0.14mg/mL was in good linear relationship with the linear regression equation y=42915x－92.541,(R 2=0.9903). Sample recovery yield was 100.1%, RSD = 1.45%, and precision of RSD = 0.96%.In comparison of separation and determination of salidroside by two methods, the results showed that both high-performance liquid chromatography and capillary electrophoresis can well work on salidroside cistanche separation without significant differences in measurements, and its content ranges from 0.120% to 0.103%.4. Study on the fingerprint of Herba Cistanch eIn this paper, HPCE method was employed to analyze the ingredient in Cistanche samples. The main active ingredient, the Acteoside of Herba Cistanch e, was set as evaluation index to establish the HPCE cistanche fingerprint, and the separation degree and reproducibility are reliable and excellent. The RSD of all relative peak area and the relative retention time are less than 5%. Our research provides a solid scientific basis to the establishment of quality standards of medicinal Cistanch e.Key words :Herba Cistanch e, polysaccharides, acteoside, salidroside, fingerprint目录摘要 (I)Abstract (III)第一章前言 (1)1．1 化学成分 (3)1.1.1 苯乙醇苷类 (3)1.1.2 多糖 (4)1.1.3 环烯醚萜类 (4)1.1.4 木质素 (5)1.1.5 挥发油成分 (5)1.1.6 其它类 (5)1.2 提取、分离纯化新技术 (6)1.2.1超临界流体萃取技术 (6)1.2.2 超声辅助提取技术 (7)1.2.3 微波辅助提取技术 (8)1.2.4 大孔树脂吸附分离技术 (9)1.2.5 高速逆流色谱分离技术 (9)1.2.6高效液相色谱技术 (10)1.2.7 高效毛细管电泳技术 (11)1.3 含量测定 (11)1.4中药指纹图谱的研究 (12)第二章肉苁蓉多糖的研究 (15)2.1 引言 (15)2.2 实验部分 (15)2.2.1 实验材料 (15)2.2.2 实验方法 (16)2.3 多糖含量测定方法的研究 (17)2.3.1 测定波长的选择 (17)2.3.2 线性关系的考察 (18)2.3.3 精密度试验 (19)2.3.4 稳定性试验 (20)2.3.5 加样回收试验 (21)2．4 结果 (22)2.4.1 计算公式 (22)2.4.2 肉苁蓉可溶性多糖和粗多糖含量的测定 (22)2.4.3 不同多糖部位的含量的测定 (22)2．5 讨论 (23)第三章高效毛细管电泳法测定肉苁蓉中毛蕊花糖苷的含量 (24)3.1 引言 (24)3.2实验部分 (24)3.2.1 实验与仪器 (24)3.3 方法学考察与结果 (25)3.3.1 检测波长的选择 (26)3.3.2 电压对分离的影响 (26)3.3.3 缓冲溶液pH值对分离的影响 (27)3.3.4 色谱条件 (28)3.3.5 标准曲线的绘制 (28)3.3.6 精密度试验 (29)3.3.7 重现性试验 (30)3.3.8 稳定性试验 (30)3.3.9 加样回收试验 (30)3.3.10 样品溶液的测定 (30)3.4 结论 (31)第四章高效毛细管电泳法测定肉苁蓉中红景天苷的含量 (32)4.1 引言 (32)4.2 实验部分 (32)4.2.1 实验材料 (33)4.2.2 实验方法 (33)4.3 方法学考察与结果 (34)4.3.1 检测波长的选择 (34)4.3.2 色谱条件 (34)4.3.3 标准曲线的绘制 (35)4.3.5 重现性试验 (36)4.3.6 稳定性试验 (36)4.3.7 加样回收试验 (36)4.3.8 样品溶液的测定 (37)4.4 结论 (38)第五章高效液相色谱法测定肉苁蓉中红景天苷的含量 (38)5.1 引言 (38)5.2 实验部分 (38)5.2.1 实验材料 (38)5.2.2 实验方法 (39)5.3 方法学考察与结果 (39)5.3.1 检测波长的选择 (39)5.3.2 色谱条件 (39)5.3.3 流动相的选择 (40)5.3.4 参照物的选择 (40)5.3.5 线性关系的考察 (40)5.3.6 精密度试验 (41)5.3.7 重现性试验 (42)5.3.8 回收率试验 (42)5.3.9 样品测定 (43)5.4 讨论 (43)5.4.1 系统适应性试验结果比较 (43)5.4.2 回收率的比较 (44)5.4.3 重现性试验的比较 (44)5.4.4 线性关系的比较 (44)第六章肉苁蓉高效毛细管电泳指纹图谱研究 (45)6.1 引言 (45)6. 2 实验材料 (46)6.2.1 仪器 (46)6.2..2 试剂与药材 (46)6.3 肉苁蓉HPCE指纹图谱的构建 (46)6.3.1 溶液的制备 (46)6.3.2 指纹图谱构建的条件 (47)6.3.3 肉苁蓉水溶性HPCE指纹图谱的建立 (47)6.4 肉苁蓉水溶性成分HPCE指纹图谱建立依据 (50)6.4.1 样品药品的来源 (50)6.4.2 样品溶液的制备 (50)6.4.3 参照品溶液的制备 (50)6.4.4 测定条件 (50)6.4.5 方法学考察 (50)6.5 结论 (41)第七章结论与展望 (42)7.1 结论 (42)7.2 存在的问题与建议 (43)参考文献 (44)致谢 (53)作者简历 (54)导师评阅表 (55)第一章前言肉苁蓉(Herba Cistanche)为列当科(Oroban-chaceae)植物肉苁蓉(Cistanchedeserticola Y．C．ia)的带鳞片的干燥肉质茎，又名苁蓉、大芸、金笋、地精[1]。