响应曲面法用于救必应等三种药材高效提取及其提取物的药理活性研究
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摘要
近年来，响应曲面法(Response surface methodology，RSM)作为一种重要的统计学试验设计广泛应用于生物过程。通过RSM可以建立连续变量三维曲面模型，对影响生物过程变量及其交互作用进行有效评价，从而确定最佳变量范围。RSM优化生物过程所需试验组数相对较少，省时省力，实验简单，预测性好，因此该方法已经成功应用到多种生物过程的优化。此外，RSM亦在中药有效成分提取研究领域应用广泛。本论文主要应用响应曲面法深入探讨其在中药五味子，救必应，大黄等三种药用植物有效成分提取中的应用，确定最佳提取变量，优化提取条件。此外，本文对传统“清热解毒”中药——救必应的的抗肿瘤和肝损伤保护作用进行初步研究。论文共分六章。
第1章主要是介绍响应曲面法的理论方法及其在中药有效成分提取中的应用研究。对深入药理活性研究的药用植物救必应的化学成分及药理活性报道做一综述。查阅文献总结概括了药用植物五味子中木脂素类成分提取工艺研究、救必应中有效成分的提取工艺研究、大黄中蒽醌类成分的提取工艺研究。
第2章运用响应曲面法（RSM）优化加速溶剂萃取（ASE）五味子中四种木脂素类成分（五味子素，五味子醇乙，五味子甲素和五味子乙素）。通过RSM建立数学模型，利用Behnken设计，获得提取条件的优化组合。从数学模型的三维响应曲面图和等高线图，以确定五味子中四种木脂素最佳提取条件如下：提取溶剂为87％的乙醇，提取温度为160℃，静态萃取时间为10分钟，提取压力为1500psi，一个提取周期的冲水量为60％。结果表明：五味子中四种木脂素的实验值14.72mg/g，结果与模型预测值较为一致。
第3章本章中利用建立超声波辅助提取（UAE）结合响应曲面法（RSM）提取救必应中紫丁香苷。通过高效液相色谱法（HPLC-UV）对紫丁香苷进行分析和定量。利用Box-Behnken design（BBD）设计对提取溶剂、提取温度和提取时间等三个主要变量进行了优化以获得最高的提取效率。结果表明：救必应中紫丁香苷的最佳条件为超声处理频率40Hz，65％乙醇作为提取溶剂，提取时间30分钟，提取温度40℃。在最优提取条件下，预测值与实验值一致。方差分析结果表明：模型的拟合度高，RSM方法可以用于优化救必应中紫丁香苷的提取纯化。
第4章在本章中，采用响应曲面法优化结合超声波辅助提取（UEA）大黄中的五种蒽醌类成分（芦荟大黄素，大黄酸，大黄素，大黄酚和大黄素甲醚）。采用高效液相色谱（HPLC-UV）对五种蒽醌类成分进行分析和定量。利用RSM对影响UEA的三个主要因素参数（提取溶剂，提取温度和提取时间）进行了优化，以期获得最高的提取效率。结果表明：最佳提取条件为84％甲醇作为溶剂，提取时间为33分钟，提取温度67℃。这些最佳条件下，实验值与预测值较为接近。结果可靠，RSM适用于超声波提取大黄中的蒽醌类成分。
第5章利用先前获得就必应提取物进行C57小鼠体内抗肿瘤作用的研究，对其抑瘤率，免疫器官指数及血清学指标进行探讨，给药14天后观察发现：各给药组对小鼠体重基本无影响，救必应高中剂量组可以显著抑制肿瘤增长，具有显著的抑瘤率；免疫学指标结果表明：救必应可以显著升高胸腺指数，降低肝指数，对免疫器官具有一定的保护作用。通过检测肝脏中ALT和AST含量，进一步明确，救必应提取物可以改善荷瘤小鼠的肝功能，与免疫组织指数结果相一致，验证中药抗肿瘤主要通过增强免疫力而发挥作用。
第6章本章利用CCl4和单次大剂量酒精诱导小鼠肝脏损伤模型，探讨救必应提取物对肝损伤小鼠的影响，比较了各给药组对肝损伤小鼠血清中ALT，AST，肝脏中SOD和MDA活性的影响。本实验结果显示，与模型组比较，救必应提取物均能降低血清中增加的ALT和AST活性及TG浓度，结果说明，救必应提取物对细胞膜有一定的保护作用。SOD是细胞内天然存在的氧自由基清除剂，可以平衡细胞内氧化和抗氧化作用，阻止氧化应激作用的发生，SOD活性的高低反映了机体清除自由基的能力。MDA是脂质过氧化反应的产物，MDA浓度的高低可以反映机体细胞膜受自由基破坏的程度。本实验结果显示，救必应给药组对SOD和MDA均有一定的作用，但对肝脏的保护作用机制有待进一步研究。
Response Surface Methodology (RSM) is a statistical experimental design used tooptimize biological process. A continuous variable surface model can be establishedwith RSM to evaluate biological process affecting factors and their interactions andthus to determine the optimum levels. The number of experimental groups required isrelative small, and considerable manpower and material resources can be saved. Thismethodology has been successfully applied to optimization of various biologicalprocesses, and has been used well in extraction of effective ingredients of traditionalChinese medicine (TCM) by virtue of its good predictability, simple experiment andhigh efficiency. This paper mainly includes an in-depth study on the application ofRSM in extraction of effective ingredients of three medicinal plants in TCM, that isChinese magnoliavine fruit, Ovateleaf Holly Bark and Rheum palmatum L., and astudy on the protection functions of Ovateleaf Holly Bark against tumor and liverinjury. This paper consists of six chapters.
Chapter one: Studies on extraction processes of lignin in Chinese magnoliavine fruit,effective ingredients in Ovateleaf Holly Bark, and anthraquinone in rheum officinatewere summarized based on literature consultation. Meanwhile, in consideration of theadvantages of RSM, studies on application of RSM for TCM effective ingredientextraction were also summarized. Besides, an overview was provided for the reportson chemical ingredients and pharmacological activity of Ovateleaf Holly Bark, forwhich in-depth pharmacological activity study was given.
Chapter two: A new method based on accelerated solvent extraction (ASE)combined with response surface methodology (RSM) modeling and optimization hasbeen developed for the extraction of four lignans in Fructus Schisandrae (the fruits ofSchisandra chinensis Baill). The RSM method, based on a three level and threevariable Box-Behnken design (BBD), was employed to obtain the optimalcombination of extraction condition. In brief, the lignans schizandrin, schisandrol B, deoxyschizandrin and schisandrin B were optimally extracted with87%ethanol asextraction solvent, extraction temperature of160°C, static extraction time of10min,extraction pressure of1,500psi, flush volume of60%and one extraction cycle. The3D response surface plot and the contour plot derived from the mathematical modelswere applied to determine the optimal conditions. Under the above conditions, theexperimental value of four lignans was14.72mg/g, which is in close agreement withthe value predicted by the model.
Chapter three: In this work, a rapid extraction method based on ultrasound-assistedextraction (UAE) of syringin from the bark of Ilex rotunda Thumb using responsesurface methodology (RSM) is described. The syringin was analyzed and quantifiedby high performance liquid chromatography coupled with UV detection (HPLC-UV).The extraction solvent, extraction temperature and extraction time, the three mainfactors for UAE, were optimized with Box-Behnken design (BBD) to obtain thehighest extraction efficiency. The optimal conditions were the use of a sonicationfrequency of40kHz,65%methanol as the solvent, an extraction time of30min andan extraction temperature of40°C. Using these optimal conditions, the experimentalvalues agreed closely with the predicted values. The analysis of variance (ANOVA)indicated a high goodness of model fit and the success of the RSM method foroptimizing syringin extraction from the bark of I. rotunda.
Chapter four: In this paper, ultrasound-assisted extraction (UAE) was applied to theextraction of anthraquinones (aloe-emodin, rhein, emodin, chrysophanol andphyscion) from Rheumpalmatum L. The five anthraquinones were quantified andanalyzed by high performance liquid chromatography coupled with UV detection(HPLC-UV). The extraction solvent,extraction temperature and extraction timeparameters, the three main factors for UAE,were optimized with response surfacemethodology (RSM) to obtain the highest extraction efficiency. The optimalconditions were the use of84%methanol as solvent, an extraction time of33min andan extraction temperature of67°C. Under these optimal conditions, the experimental values agreed closely with the predicted values. The analysis of variance indicated ahigh goodness of model fit and the success of RSM method for optimizinganthraquinones extraction in Rheum palmatum L.
Chapter five: As a traditional folk medicine, Ovateleaf Holly Bark can remarkablyclear away heat and toxic material. Modern medicine has proved that TCM with thefunction of―clearing away heat and toxic material‖can effective treat tumor, inhibitproliferation of tumor cells, and prolong life of patients. In this chapter, the authorstudies the in-vivo antitumor function of Ovateleaf Holly Bark in C57mice using theextractions of Ovateleaf Holly Bark prepared before, and explores the tumorinhibition rate, the immune organ index and the serological indexes. It was observed14days after the administration that: the administration had by and large no impact onthe weights of the mice in all experimental groups, and the tumor growth wassignificantly inhibited in the groups administered with high and middle dose ofOvateleaf Holly Bark, showing a significant tumor inhibition rate. Theimmunological indexes showed that Ovateleaf Holly Bark can significantly increasethymus index and reduce liver index, indicating considerable protection for immuneorgans. It was further verified by measurement of the ALT and AST contents in liverthat Ovateleaf Holly Bark extractions can improve liver function of tumor bearingmice, which was consistent with the results of the immune organ index. This alsoverified that antitumor TCM mainly functions through enhancement of immunity.
Chapter six: In this chapter, a model of mouse liver injury induced by CCl4andsingle administration of large dose of alcohol was used to study the impact ofOvateleaf Holly Bark extraction on mice with liver injury. The impacts of theextraction administered on the activity of ALT and ASR in serum and SOD and MDAin liver, as indicated by the experimental groups, were compared. The experimentalresults showed that, compared with the model, the increase in ALT and AST activityand TG concentration in serum was reduced by the Ovateleaf Holly Bark extraction inall experimental groups, indicating considerable protection function of Ovateleaf Holly Bark extraction for cell membranes. SOD, a scavenger of oxygen free radicalsnaturally existing in cell, can help to balance the intracellular oxidation andantioxidation and prevent oxidative stress. Level of SOD activity reflects the ability ofbody to clean out free radicals. MDA is a product of lipid peroxidation, andconcentration of MDA reflects the degree of cell membrane damage caused by freeradicals. The results of this experiment showed considerable enhancement function ofOvateleaf Holly Bark extraction for SOD and MDA. However, the mechanism of itsprotection for liver has yet to be further studied.
第1章主要是介绍响应曲面法的理论方法及其在中药有效成分提取中的应用研究。对深入药理活性研究的药用植物救必应的化学成分及药理活性报道做一综述。查阅文献总结概括了药用植物五味子中木脂素类成分提取工艺研究、救必应中有效成分的提取工艺研究、大黄中蒽醌类成分的提取工艺研究。
第2章运用响应曲面法（RSM）优化加速溶剂萃取（ASE）五味子中四种木脂素类成分（五味子素，五味子醇乙，五味子甲素和五味子乙素）。通过RSM建立数学模型，利用Behnken设计，获得提取条件的优化组合。从数学模型的三维响应曲面图和等高线图，以确定五味子中四种木脂素最佳提取条件如下：提取溶剂为87％的乙醇，提取温度为160℃，静态萃取时间为10分钟，提取压力为1500psi，一个提取周期的冲水量为60％。结果表明：五味子中四种木脂素的实验值14.72mg/g，结果与模型预测值较为一致。
第3章本章中利用建立超声波辅助提取（UAE）结合响应曲面法（RSM）提取救必应中紫丁香苷。通过高效液相色谱法（HPLC-UV）对紫丁香苷进行分析和定量。利用Box-Behnken design（BBD）设计对提取溶剂、提取温度和提取时间等三个主要变量进行了优化以获得最高的提取效率。结果表明：救必应中紫丁香苷的最佳条件为超声处理频率40Hz，65％乙醇作为提取溶剂，提取时间30分钟，提取温度40℃。在最优提取条件下，预测值与实验值一致。方差分析结果表明：模型的拟合度高，RSM方法可以用于优化救必应中紫丁香苷的提取纯化。
第4章在本章中，采用响应曲面法优化结合超声波辅助提取（UEA）大黄中的五种蒽醌类成分（芦荟大黄素，大黄酸，大黄素，大黄酚和大黄素甲醚）。采用高效液相色谱（HPLC-UV）对五种蒽醌类成分进行分析和定量。利用RSM对影响UEA的三个主要因素参数（提取溶剂，提取温度和提取时间）进行了优化，以期获得最高的提取效率。结果表明：最佳提取条件为84％甲醇作为溶剂，提取时间为33分钟，提取温度67℃。这些最佳条件下，实验值与预测值较为接近。结果可靠，RSM适用于超声波提取大黄中的蒽醌类成分。
第5章利用先前获得就必应提取物进行C57小鼠体内抗肿瘤作用的研究，对其抑瘤率，免疫器官指数及血清学指标进行探讨，给药14天后观察发现：各给药组对小鼠体重基本无影响，救必应高中剂量组可以显著抑制肿瘤增长，具有显著的抑瘤率；免疫学指标结果表明：救必应可以显著升高胸腺指数，降低肝指数，对免疫器官具有一定的保护作用。通过检测肝脏中ALT和AST含量，进一步明确，救必应提取物可以改善荷瘤小鼠的肝功能，与免疫组织指数结果相一致，验证中药抗肿瘤主要通过增强免疫力而发挥作用。
第6章本章利用CCl4和单次大剂量酒精诱导小鼠肝脏损伤模型，探讨救必应提取物对肝损伤小鼠的影响，比较了各给药组对肝损伤小鼠血清中ALT，AST，肝脏中SOD和MDA活性的影响。本实验结果显示，与模型组比较，救必应提取物均能降低血清中增加的ALT和AST活性及TG浓度，结果说明，救必应提取物对细胞膜有一定的保护作用。SOD是细胞内天然存在的氧自由基清除剂，可以平衡细胞内氧化和抗氧化作用，阻止氧化应激作用的发生，SOD活性的高低反映了机体清除自由基的能力。MDA是脂质过氧化反应的产物，MDA浓度的高低可以反映机体细胞膜受自由基破坏的程度。本实验结果显示，救必应给药组对SOD和MDA均有一定的作用，但对肝脏的保护作用机制有待进一步研究。
Response Surface Methodology (RSM) is a statistical experimental design used tooptimize biological process. A continuous variable surface model can be establishedwith RSM to evaluate biological process affecting factors and their interactions andthus to determine the optimum levels. The number of experimental groups required isrelative small, and considerable manpower and material resources can be saved. Thismethodology has been successfully applied to optimization of various biologicalprocesses, and has been used well in extraction of effective ingredients of traditionalChinese medicine (TCM) by virtue of its good predictability, simple experiment andhigh efficiency. This paper mainly includes an in-depth study on the application ofRSM in extraction of effective ingredients of three medicinal plants in TCM, that isChinese magnoliavine fruit, Ovateleaf Holly Bark and Rheum palmatum L., and astudy on the protection functions of Ovateleaf Holly Bark against tumor and liverinjury. This paper consists of six chapters.
Chapter one: Studies on extraction processes of lignin in Chinese magnoliavine fruit,effective ingredients in Ovateleaf Holly Bark, and anthraquinone in rheum officinatewere summarized based on literature consultation. Meanwhile, in consideration of theadvantages of RSM, studies on application of RSM for TCM effective ingredientextraction were also summarized. Besides, an overview was provided for the reportson chemical ingredients and pharmacological activity of Ovateleaf Holly Bark, forwhich in-depth pharmacological activity study was given.
Chapter two: A new method based on accelerated solvent extraction (ASE)combined with response surface methodology (RSM) modeling and optimization hasbeen developed for the extraction of four lignans in Fructus Schisandrae (the fruits ofSchisandra chinensis Baill). The RSM method, based on a three level and threevariable Box-Behnken design (BBD), was employed to obtain the optimalcombination of extraction condition. In brief, the lignans schizandrin, schisandrol B, deoxyschizandrin and schisandrin B were optimally extracted with87%ethanol asextraction solvent, extraction temperature of160°C, static extraction time of10min,extraction pressure of1,500psi, flush volume of60%and one extraction cycle. The3D response surface plot and the contour plot derived from the mathematical modelswere applied to determine the optimal conditions. Under the above conditions, theexperimental value of four lignans was14.72mg/g, which is in close agreement withthe value predicted by the model.
Chapter three: In this work, a rapid extraction method based on ultrasound-assistedextraction (UAE) of syringin from the bark of Ilex rotunda Thumb using responsesurface methodology (RSM) is described. The syringin was analyzed and quantifiedby high performance liquid chromatography coupled with UV detection (HPLC-UV).The extraction solvent, extraction temperature and extraction time, the three mainfactors for UAE, were optimized with Box-Behnken design (BBD) to obtain thehighest extraction efficiency. The optimal conditions were the use of a sonicationfrequency of40kHz,65%methanol as the solvent, an extraction time of30min andan extraction temperature of40°C. Using these optimal conditions, the experimentalvalues agreed closely with the predicted values. The analysis of variance (ANOVA)indicated a high goodness of model fit and the success of the RSM method foroptimizing syringin extraction from the bark of I. rotunda.
Chapter four: In this paper, ultrasound-assisted extraction (UAE) was applied to theextraction of anthraquinones (aloe-emodin, rhein, emodin, chrysophanol andphyscion) from Rheumpalmatum L. The five anthraquinones were quantified andanalyzed by high performance liquid chromatography coupled with UV detection(HPLC-UV). The extraction solvent,extraction temperature and extraction timeparameters, the three main factors for UAE,were optimized with response surfacemethodology (RSM) to obtain the highest extraction efficiency. The optimalconditions were the use of84%methanol as solvent, an extraction time of33min andan extraction temperature of67°C. Under these optimal conditions, the experimental values agreed closely with the predicted values. The analysis of variance indicated ahigh goodness of model fit and the success of RSM method for optimizinganthraquinones extraction in Rheum palmatum L.
Chapter five: As a traditional folk medicine, Ovateleaf Holly Bark can remarkablyclear away heat and toxic material. Modern medicine has proved that TCM with thefunction of―clearing away heat and toxic material‖can effective treat tumor, inhibitproliferation of tumor cells, and prolong life of patients. In this chapter, the authorstudies the in-vivo antitumor function of Ovateleaf Holly Bark in C57mice using theextractions of Ovateleaf Holly Bark prepared before, and explores the tumorinhibition rate, the immune organ index and the serological indexes. It was observed14days after the administration that: the administration had by and large no impact onthe weights of the mice in all experimental groups, and the tumor growth wassignificantly inhibited in the groups administered with high and middle dose ofOvateleaf Holly Bark, showing a significant tumor inhibition rate. Theimmunological indexes showed that Ovateleaf Holly Bark can significantly increasethymus index and reduce liver index, indicating considerable protection for immuneorgans. It was further verified by measurement of the ALT and AST contents in liverthat Ovateleaf Holly Bark extractions can improve liver function of tumor bearingmice, which was consistent with the results of the immune organ index. This alsoverified that antitumor TCM mainly functions through enhancement of immunity.
Chapter six: In this chapter, a model of mouse liver injury induced by CCl4andsingle administration of large dose of alcohol was used to study the impact ofOvateleaf Holly Bark extraction on mice with liver injury. The impacts of theextraction administered on the activity of ALT and ASR in serum and SOD and MDAin liver, as indicated by the experimental groups, were compared. The experimentalresults showed that, compared with the model, the increase in ALT and AST activityand TG concentration in serum was reduced by the Ovateleaf Holly Bark extraction inall experimental groups, indicating considerable protection function of Ovateleaf Holly Bark extraction for cell membranes. SOD, a scavenger of oxygen free radicalsnaturally existing in cell, can help to balance the intracellular oxidation andantioxidation and prevent oxidative stress. Level of SOD activity reflects the ability ofbody to clean out free radicals. MDA is a product of lipid peroxidation, andconcentration of MDA reflects the degree of cell membrane damage caused by freeradicals. The results of this experiment showed considerable enhancement function ofOvateleaf Holly Bark extraction for SOD and MDA. However, the mechanism of itsprotection for liver has yet to be further studied.
引文
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