厚朴有效成分分离及中药对活性氧清除率测定方法研究
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摘要
本文建立了测定中药对生物体中三种常见活性氧(羟自由基、超氧阴离子自由基和过氧化氢)清除率的新方法，为筛选可清除这三种活性氧的物质研究提供了一种便捷的途径；另外，对目前毛细管电泳领域中十分活跃的非水毛细管电泳的分离影响因素进行了研究，在此基础之上建立了厚朴酚与和厚朴酚的非水毛细管电泳分离新方法。
弱荧光物质苯甲酸可以和·OH反应生成强荧光产物。中药提取物可以清除反应体系中的·OH，使产物的生成量减少，从而使溶液的荧光增加程度降低。据此原理建立了一种测定中药对·OH清除率的新方法。研究表明，在紫外光照射H_2O_2的·OH产生体系中，当照射时间为20min时，·OH产生率达到饱和；当H_2O_2与苯甲酸的摩尔比为30:1时，·OH可与苯甲酸完全反应；当体系中的苯甲酸浓度为8.5mmol/L时，产物荧光响应随H_2O_2浓度变化的线性范围是2.2～80mmol/L。测得厚朴和山茱萸清除·OH的IC_(50)分别为1.025mg/L和515.3mg/L，与分光光度法测定结果的一致性较好。
黄嘌呤氧化酶可催化黄嘌吟反应生成尿酸，同时产生O_2。O_2可以氧化四氮唑蓝(NBT)生成甲臜，中药提取物可以清除溶液中的O_2，使甲臜的生成速度降低。据此原理，利用动力学分光光度法建立了一种测定中药对超氧阴离子清除率的新方法。研究了反应时间对甲臜吸光度和反应速度的影响，指出灵敏测定时段在反应时间为2—7min之间。本法测得的抗坏血酸对O_2的清除率与文献报道值一致。测得厚朴和杜仲对O_2的IC_(50)分别为7.580mg/L和323.8mg/L。
基于苯胺可以被H_2O_2氧化为硝基苯，从而使其荧光猝灭的原理建立了一种测定中药对H_2O_2清除率的新方法。研究表明：在50mmol/L磷酸盐缓冲液中，当pH为7.6时，苯胺荧光强度接近于最大值，且与人体体液pH一致；而当乙醇浓度为50％(V/V)时，散射光干扰达到最小值，荧光强度达到最大值。当苯胺浓度为
11O22o！几，N2O2的浓度小于1mmd几时，苯胺荧光的粹灭程度＼H贝；浓度Y线性
关系。在此条件下测定了厚朴和杜仲对HZOZ的清除率，结双与鲁术诺化学发光法测
定值一致。
建立了中药厚朴中主要活性氧清除剂——厚朴酚与和厚朴酚的1卜水毛细管屯泳
分离分析方法。对非水溶剂的选择、不同酸根阴离子的影响、添加hlJ的选择及加量
和p＊“的影响进行了研究，确定非水介质为：乙睛、甲酞胺、甲醇的混合比例力12：！0c。
V：V：V），缓冲介质是 60mmoU Tris，水的添加浓度为 5％，叫醋酸调u pH”为 8且 6。
在该条件下对中药厚朴中的的厚朴酚与和厚朴酚进行了分离。厚朴酚与和厚朴酚的
＿工作曲线sj别y］ Y＝7685．56＋11388265．36X 和 Y＝23744．94＋17697231．73X，相关系数
分别达到 0．9996和 0．9998。
Primary products storage and process engineering TIAN Yi-ling Abstract: The methods of eliminating three kinds of oxygen radicals (hydroxyl radical, superoxied anion and hydrogen peroxide) which can accelerate senile process were developed. They provide a simple path to choose the substances eliminating the oxygen radicals. It was also studied factors how to influence separation in nonaqueous capillary electrophoresis which is very lively in capillary electrophoresis. And a new method of separating magnolol and honokiol in magnolia bark was developed on the base.
Benzoic acid with weak fluorescence may reacted on 'OH, and products with intense fluorescence are made. Extractives of Chinese traditional medicine may eliminate ?OH in solution, and make amounts of the products to reduce. Then, increase level of fluorescence of products in solution will be lowered. Based on this principle, a new method is developed to determine the IC50 of Chinese traditional medicine to ?OH. It is shown that productivity of ?OH tends to saturation when HiO: is shone more than 20 minutes by 280nm UV ray; ?OH may react on benzole acid completely when molar ratio of H2O2 to benzoic acid is 30:1; linear response range of products fluorescence is 2.2~80 mmol/L with concentration of H2O2. IC50 to eliminate OH with magnoliae and eucommia are 1.025mg/L and 515.3mg/L respectively. There are no remarkable difference between these results and that of spectrophotometry.
Xanthine oxidase(XO) may catalyze the reaction of xanthine gaving uric acid and superoxide anion Cy") ?O2~can oxidize NET to form formazan. Extractives of Chinese traditional medicine may eliminate O2V in solution, and decrease the production velocity
-51 -
of formazan. A simple and sensitive kinetic spectrophotometric method to determination the ICso of Chinese traditional medicine to O2* is reported based on this principle. The effects of reaction time on absorbance and reaction velocity were studied. It is shown that sensitivity is higher from 1 min tol min. IC50 of vitamin C to O2vwas determined and compared with literature. The results are identical. ICso of Cortex Magnolias officinalis and Cortex Eucommia to CV are determined.
Aniline may be oxidized to be nitrobenzene and its fluorescence quenched by h^O?. Based on this principle, a new method is developed to determine the 1C50 of Chinese traditional medicine to t^Cb. It is shown that fluorescence of aniline is close to maximum when pH of 50mmol/L phosphate buffer solution is 7.6, and this pH is consist with that of human blood. Scatter interference is minimum and fluorescence is maximum when concentration of ethanol is 50%(v/v). Linear relation is between quenching level of aniline fluorescence and concentration of H:O2 when aniline is HOmmol/L and H:C>2 is below Immol/L. The 1C50 of Cortex Magnolias officinalis and Cortex Eucommia to HUC^ are determined in the condition mentioned above. The results are identical with that of luminol chemiluminescence.
A method was developed to separate magnolol and honokiol in Chinese tradition medicine Magnolia officinalis by nonaqueous capillary electrophoresis, which are principal elimination components to active oxygen species. It was studied how to choose nonaqueouse solvents, additive, and pH* and acid radical anion how to influence separation. The ratio of Acetonitrile to formamide to methanol is 12: 10: 3 (V: V: V), background electrolyte is 60 mmoI/L tris, and the concentration of water is 5%. The pH* of nonaqueouse buffer was adjusted by acetic acid. Magnolol and honokiol were separated under these conditions. The calibration curves were ?7685.56+11388265.36X and ?23744.94+17697231.73X, and correlation coefficients were 0.9996 and 0.9998 for
-52-
magnolol and honokiol respectively.
弱荧光物质苯甲酸可以和·OH反应生成强荧光产物。中药提取物可以清除反应体系中的·OH，使产物的生成量减少，从而使溶液的荧光增加程度降低。据此原理建立了一种测定中药对·OH清除率的新方法。研究表明，在紫外光照射H_2O_2的·OH产生体系中，当照射时间为20min时，·OH产生率达到饱和；当H_2O_2与苯甲酸的摩尔比为30:1时，·OH可与苯甲酸完全反应；当体系中的苯甲酸浓度为8.5mmol/L时，产物荧光响应随H_2O_2浓度变化的线性范围是2.2～80mmol/L。测得厚朴和山茱萸清除·OH的IC_(50)分别为1.025mg/L和515.3mg/L，与分光光度法测定结果的一致性较好。
黄嘌呤氧化酶可催化黄嘌吟反应生成尿酸，同时产生O_2。O_2可以氧化四氮唑蓝(NBT)生成甲臜，中药提取物可以清除溶液中的O_2，使甲臜的生成速度降低。据此原理，利用动力学分光光度法建立了一种测定中药对超氧阴离子清除率的新方法。研究了反应时间对甲臜吸光度和反应速度的影响，指出灵敏测定时段在反应时间为2—7min之间。本法测得的抗坏血酸对O_2的清除率与文献报道值一致。测得厚朴和杜仲对O_2的IC_(50)分别为7.580mg/L和323.8mg/L。
基于苯胺可以被H_2O_2氧化为硝基苯，从而使其荧光猝灭的原理建立了一种测定中药对H_2O_2清除率的新方法。研究表明：在50mmol/L磷酸盐缓冲液中，当pH为7.6时，苯胺荧光强度接近于最大值，且与人体体液pH一致；而当乙醇浓度为50％(V/V)时，散射光干扰达到最小值，荧光强度达到最大值。当苯胺浓度为
11O22o！几，N2O2的浓度小于1mmd几时，苯胺荧光的粹灭程度＼H贝；浓度Y线性
关系。在此条件下测定了厚朴和杜仲对HZOZ的清除率，结双与鲁术诺化学发光法测
定值一致。
建立了中药厚朴中主要活性氧清除剂——厚朴酚与和厚朴酚的1卜水毛细管屯泳
分离分析方法。对非水溶剂的选择、不同酸根阴离子的影响、添加hlJ的选择及加量
和p＊“的影响进行了研究，确定非水介质为：乙睛、甲酞胺、甲醇的混合比例力12：！0c。
V：V：V），缓冲介质是 60mmoU Tris，水的添加浓度为 5％，叫醋酸调u pH”为 8且 6。
在该条件下对中药厚朴中的的厚朴酚与和厚朴酚进行了分离。厚朴酚与和厚朴酚的
＿工作曲线sj别y］ Y＝7685．56＋11388265．36X 和 Y＝23744．94＋17697231．73X，相关系数
分别达到 0．9996和 0．9998。
Primary products storage and process engineering TIAN Yi-ling Abstract: The methods of eliminating three kinds of oxygen radicals (hydroxyl radical, superoxied anion and hydrogen peroxide) which can accelerate senile process were developed. They provide a simple path to choose the substances eliminating the oxygen radicals. It was also studied factors how to influence separation in nonaqueous capillary electrophoresis which is very lively in capillary electrophoresis. And a new method of separating magnolol and honokiol in magnolia bark was developed on the base.
Benzoic acid with weak fluorescence may reacted on 'OH, and products with intense fluorescence are made. Extractives of Chinese traditional medicine may eliminate ?OH in solution, and make amounts of the products to reduce. Then, increase level of fluorescence of products in solution will be lowered. Based on this principle, a new method is developed to determine the IC50 of Chinese traditional medicine to ?OH. It is shown that productivity of ?OH tends to saturation when HiO: is shone more than 20 minutes by 280nm UV ray; ?OH may react on benzole acid completely when molar ratio of H2O2 to benzoic acid is 30:1; linear response range of products fluorescence is 2.2~80 mmol/L with concentration of H2O2. IC50 to eliminate OH with magnoliae and eucommia are 1.025mg/L and 515.3mg/L respectively. There are no remarkable difference between these results and that of spectrophotometry.
Xanthine oxidase(XO) may catalyze the reaction of xanthine gaving uric acid and superoxide anion Cy") ?O2~can oxidize NET to form formazan. Extractives of Chinese traditional medicine may eliminate O2V in solution, and decrease the production velocity
-51 -
of formazan. A simple and sensitive kinetic spectrophotometric method to determination the ICso of Chinese traditional medicine to O2* is reported based on this principle. The effects of reaction time on absorbance and reaction velocity were studied. It is shown that sensitivity is higher from 1 min tol min. IC50 of vitamin C to O2vwas determined and compared with literature. The results are identical. ICso of Cortex Magnolias officinalis and Cortex Eucommia to CV are determined.
Aniline may be oxidized to be nitrobenzene and its fluorescence quenched by h^O?. Based on this principle, a new method is developed to determine the 1C50 of Chinese traditional medicine to t^Cb. It is shown that fluorescence of aniline is close to maximum when pH of 50mmol/L phosphate buffer solution is 7.6, and this pH is consist with that of human blood. Scatter interference is minimum and fluorescence is maximum when concentration of ethanol is 50%(v/v). Linear relation is between quenching level of aniline fluorescence and concentration of H:O2 when aniline is HOmmol/L and H:C>2 is below Immol/L. The 1C50 of Cortex Magnolias officinalis and Cortex Eucommia to HUC^ are determined in the condition mentioned above. The results are identical with that of luminol chemiluminescence.
A method was developed to separate magnolol and honokiol in Chinese tradition medicine Magnolia officinalis by nonaqueous capillary electrophoresis, which are principal elimination components to active oxygen species. It was studied how to choose nonaqueouse solvents, additive, and pH* and acid radical anion how to influence separation. The ratio of Acetonitrile to formamide to methanol is 12: 10: 3 (V: V: V), background electrolyte is 60 mmoI/L tris, and the concentration of water is 5%. The pH* of nonaqueouse buffer was adjusted by acetic acid. Magnolol and honokiol were separated under these conditions. The calibration curves were ?7685.56+11388265.36X and ?23744.94+17697231.73X, and correlation coefficients were 0.9996 and 0.9998 for
-52-
magnolol and honokiol respectively.
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