苎麻叶和红豆杉中活性成分的提取、纯化及测定研究
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摘要
本文研究了苎麻叶中活性成分—绿原酸及红豆杉(Taxus chinensis(Pilger)Rehd)中活性成分—紫杉醇的提取、纯化及测定方法。
(1)确定了以乙醇水溶液提取绿原酸和紫杉醇的最优条件。使用pH=4.0的70％的乙醇溶液在70℃下浸提苎麻叶三次，每次浸提时间2h，料液比为1：10，绿原酸的浸提率达83％；用85％乙醇以1：10的料液比在75℃下浸提两次，每次2h，紫杉醇的提取率为90.24％。
(2)分别探讨了流动相、酸种类等因素对高效液相色谱(HPLC)分析绿原酸和紫杉醇含量的影响。确定了绿原酸的最佳色谱条件：ODS色谱柱(200×4.6 mm，5μm)，18％甲醇+1.0％的乙酸为流动相，流速1.0mL／min，检测波长326nm，进样体积6μL，分析时间为14 min。该方法校准曲线为Y=1.618×10~6X-23081.3(r=0.9997)，线性范围为20mg／L-160mg／L，方法的加标回收率为97％—101％，绿原酸与相邻杂质的分离度为1.55，该方法可满足分析要求，测得苎麻叶中含绿原酸0.52％；紫杉醇的最佳测定条件为：λ=227nm，ODS色谱柱(200×4.6 mm，5μm)，甲醇：水：乙酸(v／v)=55：44：1为流动相，流速1.0mL／min，进样浓度在0.05mg／mL—0.5mg／mL范围内，分析时间为28 min，该方法校准曲线为y=13021.7+1.01×10~6x(r=0.9990)，方法的加标回收率为96％—104％。
(3)采用比色法测定树脂纯化部分流出液中绿原酸的含量。在最佳比色及检测条件下拟定的操作程序如下：取适量溶液于5mL比色管中，加热挥去甲醇，加入1.00mL水，0.100mL 12.5％(v／v)的醋酸水溶液，2.00mL 7％(w／w)的尿素水溶液，0.250mL 0.5％(w／w)的亚硝酸钠水溶液混合均匀，3min后加入0.500mL 5％(w／w)的氢氧化钠水溶液后定容至刻度。比色，以水为空白，1h之内测量体系在510nm处的吸光光度值。该方法标准曲线为A=0.037+0.1602X(r=0.9998)，线性范围为0.05mg-0.6mg，方法的加标回收率为95％—107％，测
硕士学位论文
摘要
定绿原酸浓度的RSD二2 .4%。
(4)研究了层析法处理芒麻叶浸提液和红豆杉浸提液的工艺条件。
采用AB一8做柱填料，采用15 x 400Inln层析柱，试验装置简单，
吸附及洗脱易于进行，试验中采用了无毒无污染的乙醇溶液，操作
安全方便。通过试验确定上柱样为pH=4 .0含绿原酸1.4mg/mL的溶
液(加入4%的NaCI，w/v)，吸附流速为1 .SBV/h，且上柱样重复过
柱一次，当吸附时间达到30min时用pH=9 .0的30%乙醇溶液进行洗
脱，洗脱剂流速为 1.OBV/h，洗脱率达95%，AB一8对绿原酸的选择
性好，通过吸附一脱附试验确定树脂分离绿原酸的最大吸附量为
23.Slng/mL树脂，可使产品中绿原酸的含量由3.53%提高到41.31
%。
采用碱性氧化铝作柱填料，层析柱柱尺寸为:巧IlunX250Inln，通
过试验确定最佳层析条件为:物料在柱中的反应时间为30min，2.0:
98.0(V/V)的甲醇/三氧甲烷为平衡液，4.0:96.0(V/V)的甲醇/
三氯甲烷为洗脱液，经一步氧化铝层析，可使产品中紫杉醇的含量
由1.87%提高到16.59%，紫杉醇的回收率接近170%。
The extraction, purification and analysis method of the biologically active composition chlorogenic acid in ramie leaf and paclitaxel in Taxus were discussed.
(1) Technological extraction process of chlorogenic and paclitaxel with organic solvent was experimented.
The solvent of 70% ethanol aqueous was used to extract from ramie leaf three times for a duration of two hours, ratio of feed-stock weight to the volume of solvent is 1:10. Temperature is 70℃ and pH is 4.0, the extraction ratio was 83%.
The extraction ratio of paclitaxel was 90.24% when extracted with 1:10 of 85% ethanol aqueous, twice at 75 ℃ and 2 hours.
(2) Effects of mobile phase, acid and velocity on the analysis of chlorogenic acid and paclitaxel were discussed as well.
The content of chlorogenic acid in ramie leaf were determined by HPLC and the analysis condition of chlorogenic acid was as follows: ODS column (200x4.6 mm, 5 um), 18% methanol aqueous and 1.0% acetic acid as mobile phase, UV detector at 326 nm, injection volume was 6 U L, the experimental separating time was 14 min. The calibration curve was Y=1.618 X 106X-23081.3 (r=0.9997) and the linear determination range is 20mg/L-160mg/L, the recovery was 97% -101 %. The method could meet the requirement of quickly analysis and various results; the content of chlorogenic acid is 0.52%.
The analysis condition of paclitaxel was as follows: methanol: watenacetic acid=55:44:1 (v/v), UV detector at 221 nm, the experimental separating was 28min and the calibration cure was y = 13021.7 + 1.01 X 106x (r=0.9990) which was linear in the concentration range of 0.05mg/mL-0.5mg/mL and the recovery was 96% -104%.
(3) A method for the colorimetry of chlorogenic acid was studied. The optimal coloration and examining conditions were as follows: appropriative sample was added in the colorimetric tube, then evaporated methanol, added 1.00mL H2O, 0.100mL acetic acid aqueous, 2.00mL urea aqueous and 0.250mL sodium nitrite aqueous, blended
them evenly, 0.500mL sodium hydroxide aqueous tree minutes later. The adsorption value was measured at 510nm within one hour with water as consultation. The calibration curve was A=0.037+0.1602X (r=0.9998) and the linear is in the range of 0.05mg-0.6mg, the recovery was 95% - 107%, the RSD of the content of chlorogenic acid was 2.4%. (4) Technological process of purification for chlorogenic acid and paclitaxel were completed by chromatographic column.
Employed with 15X400 mm column, the installation was simple and the adsorption and desorption was easy to operate. In this experiment, the ethanol was used which is no pollution and no poison. Chlorogenic acid was absorbed well by AB-8 resin. The condition obtained was as follows: The content of chlorogenic acid in injection sample is 1.4mg/mL and pH is 4.0 (4% Nacl was added, w/v) at the flow rate 1.5BV/h, the injection sample must be added again and the adsorption time is 30min, then eluted by 30% ethanol aqueous which pH is 9.0 at the rate 1.0BV/h, the desorption ratio was 95% and the optimum adsorption amount by chlorogenic acid adsorption-desorption experiment was 23.8mg/mL. The purity of chlorogenic acid reached 41.31% from 3.53%.
The Al2O3 chromatographic process established the conversion and separation of paclitaxel from Taxus, the column is 15 X 250 mm while the reaction time is 30min, the balancing liquid is 2% CH3OH and 98% CHCl3 and the eluent compositions is 4% CH3OH and 96% CHCl3. The purity of paclitaxel reached 16.59% from 1.87% with the recovery of 170% after the one chromatographic step.
(1)确定了以乙醇水溶液提取绿原酸和紫杉醇的最优条件。使用pH=4.0的70％的乙醇溶液在70℃下浸提苎麻叶三次，每次浸提时间2h，料液比为1：10，绿原酸的浸提率达83％；用85％乙醇以1：10的料液比在75℃下浸提两次，每次2h，紫杉醇的提取率为90.24％。
(2)分别探讨了流动相、酸种类等因素对高效液相色谱(HPLC)分析绿原酸和紫杉醇含量的影响。确定了绿原酸的最佳色谱条件：ODS色谱柱(200×4.6 mm，5μm)，18％甲醇+1.0％的乙酸为流动相，流速1.0mL／min，检测波长326nm，进样体积6μL，分析时间为14 min。该方法校准曲线为Y=1.618×10~6X-23081.3(r=0.9997)，线性范围为20mg／L-160mg／L，方法的加标回收率为97％—101％，绿原酸与相邻杂质的分离度为1.55，该方法可满足分析要求，测得苎麻叶中含绿原酸0.52％；紫杉醇的最佳测定条件为：λ=227nm，ODS色谱柱(200×4.6 mm，5μm)，甲醇：水：乙酸(v／v)=55：44：1为流动相，流速1.0mL／min，进样浓度在0.05mg／mL—0.5mg／mL范围内，分析时间为28 min，该方法校准曲线为y=13021.7+1.01×10~6x(r=0.9990)，方法的加标回收率为96％—104％。
(3)采用比色法测定树脂纯化部分流出液中绿原酸的含量。在最佳比色及检测条件下拟定的操作程序如下：取适量溶液于5mL比色管中，加热挥去甲醇，加入1.00mL水，0.100mL 12.5％(v／v)的醋酸水溶液，2.00mL 7％(w／w)的尿素水溶液，0.250mL 0.5％(w／w)的亚硝酸钠水溶液混合均匀，3min后加入0.500mL 5％(w／w)的氢氧化钠水溶液后定容至刻度。比色，以水为空白，1h之内测量体系在510nm处的吸光光度值。该方法标准曲线为A=0.037+0.1602X(r=0.9998)，线性范围为0.05mg-0.6mg，方法的加标回收率为95％—107％，测
硕士学位论文
摘要
定绿原酸浓度的RSD二2 .4%。
(4)研究了层析法处理芒麻叶浸提液和红豆杉浸提液的工艺条件。
采用AB一8做柱填料，采用15 x 400Inln层析柱，试验装置简单，
吸附及洗脱易于进行，试验中采用了无毒无污染的乙醇溶液，操作
安全方便。通过试验确定上柱样为pH=4 .0含绿原酸1.4mg/mL的溶
液(加入4%的NaCI，w/v)，吸附流速为1 .SBV/h，且上柱样重复过
柱一次，当吸附时间达到30min时用pH=9 .0的30%乙醇溶液进行洗
脱，洗脱剂流速为 1.OBV/h，洗脱率达95%，AB一8对绿原酸的选择
性好，通过吸附一脱附试验确定树脂分离绿原酸的最大吸附量为
23.Slng/mL树脂，可使产品中绿原酸的含量由3.53%提高到41.31
%。
采用碱性氧化铝作柱填料，层析柱柱尺寸为:巧IlunX250Inln，通
过试验确定最佳层析条件为:物料在柱中的反应时间为30min，2.0:
98.0(V/V)的甲醇/三氧甲烷为平衡液，4.0:96.0(V/V)的甲醇/
三氯甲烷为洗脱液，经一步氧化铝层析，可使产品中紫杉醇的含量
由1.87%提高到16.59%，紫杉醇的回收率接近170%。
The extraction, purification and analysis method of the biologically active composition chlorogenic acid in ramie leaf and paclitaxel in Taxus were discussed.
(1) Technological extraction process of chlorogenic and paclitaxel with organic solvent was experimented.
The solvent of 70% ethanol aqueous was used to extract from ramie leaf three times for a duration of two hours, ratio of feed-stock weight to the volume of solvent is 1:10. Temperature is 70℃ and pH is 4.0, the extraction ratio was 83%.
The extraction ratio of paclitaxel was 90.24% when extracted with 1:10 of 85% ethanol aqueous, twice at 75 ℃ and 2 hours.
(2) Effects of mobile phase, acid and velocity on the analysis of chlorogenic acid and paclitaxel were discussed as well.
The content of chlorogenic acid in ramie leaf were determined by HPLC and the analysis condition of chlorogenic acid was as follows: ODS column (200x4.6 mm, 5 um), 18% methanol aqueous and 1.0% acetic acid as mobile phase, UV detector at 326 nm, injection volume was 6 U L, the experimental separating time was 14 min. The calibration curve was Y=1.618 X 106X-23081.3 (r=0.9997) and the linear determination range is 20mg/L-160mg/L, the recovery was 97% -101 %. The method could meet the requirement of quickly analysis and various results; the content of chlorogenic acid is 0.52%.
The analysis condition of paclitaxel was as follows: methanol: watenacetic acid=55:44:1 (v/v), UV detector at 221 nm, the experimental separating was 28min and the calibration cure was y = 13021.7 + 1.01 X 106x (r=0.9990) which was linear in the concentration range of 0.05mg/mL-0.5mg/mL and the recovery was 96% -104%.
(3) A method for the colorimetry of chlorogenic acid was studied. The optimal coloration and examining conditions were as follows: appropriative sample was added in the colorimetric tube, then evaporated methanol, added 1.00mL H2O, 0.100mL acetic acid aqueous, 2.00mL urea aqueous and 0.250mL sodium nitrite aqueous, blended
them evenly, 0.500mL sodium hydroxide aqueous tree minutes later. The adsorption value was measured at 510nm within one hour with water as consultation. The calibration curve was A=0.037+0.1602X (r=0.9998) and the linear is in the range of 0.05mg-0.6mg, the recovery was 95% - 107%, the RSD of the content of chlorogenic acid was 2.4%. (4) Technological process of purification for chlorogenic acid and paclitaxel were completed by chromatographic column.
Employed with 15X400 mm column, the installation was simple and the adsorption and desorption was easy to operate. In this experiment, the ethanol was used which is no pollution and no poison. Chlorogenic acid was absorbed well by AB-8 resin. The condition obtained was as follows: The content of chlorogenic acid in injection sample is 1.4mg/mL and pH is 4.0 (4% Nacl was added, w/v) at the flow rate 1.5BV/h, the injection sample must be added again and the adsorption time is 30min, then eluted by 30% ethanol aqueous which pH is 9.0 at the rate 1.0BV/h, the desorption ratio was 95% and the optimum adsorption amount by chlorogenic acid adsorption-desorption experiment was 23.8mg/mL. The purity of chlorogenic acid reached 41.31% from 3.53%.
The Al2O3 chromatographic process established the conversion and separation of paclitaxel from Taxus, the column is 15 X 250 mm while the reaction time is 30min, the balancing liquid is 2% CH3OH and 98% CHCl3 and the eluent compositions is 4% CH3OH and 96% CHCl3. The purity of paclitaxel reached 16.59% from 1.87% with the recovery of 170% after the one chromatographic step.
引文
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