| 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. |
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