【作者】 李永生；

【导师】 田平芳；

【作者基本信息】 北京化工大学 ， 制药工程， 2009， 硕士

【摘要】 进入21世纪以来,随着化石资源与能源短缺、环境危机日益加剧,生物催化技术作为绿色化学与绿色化工发展的重要手段,其发展必将对医药、能源、农业、食品、生命科学等领域产生巨大影响。利用生物酶催化技术提取和分离天然产物中的有效成分是一项新兴技术。在天然产物有效成分提取过程中,活性成分通常被植物纤维所包裹,植物细胞壁是提取有效成分的主要屏障,使有效成分不能很好的与反应体系接触,阻碍细胞中有效成分的溶出。选用恰当的酶,通过酶催化反应温和地水解或降解植物细胞壁成分,可加速有效成分的溶出,提高了目标产物的提取率,降低了成本。目前,国内外对纤维素酶辅助提取乌头生物碱的研究没有文献报道,本研究初步探索纤维素酶催化转化乌头生物碱的反应条件和反应机制。本研究以乌头碱和乌头总碱的提取率为指标,先通过单因素实验考察纤维素酶催化对乌头生物碱提取率的影响,确定纤维素酶催化反应的温度、pH值、纤维素酶用量、作用时间等因素的范围;再通过正交试验确定纤维素酶催化转化乌头生物碱的最佳条件。并与现有的乌头生物碱提取方法:酸水提取法、大孔吸附树脂提取法、半仿生提取法、乙醇提取法作了对比实验。实验结果表明:利用纤维素酶催化转化乌头生物碱时,纤维素酶的适宜作用条件为温度:45℃,pH值:4.5,酶用量:8mg/g(1 g药材中加入0.08 g纤维素酶),作用时间:4.0 h,乌头碱和乌头总碱的提取率分别为0.002447%和0.244410%。与酸水提取法比较,酶法提取的乌头碱、乌头总碱的提取率分别提高84.4%和59.3%;与大孔吸附树脂提取比较,酶法提取的乌头碱、乌头总碱分别提高61.1%和41.4%;与半仿生提取比较,酶法提取的乌头碱、乌头总碱提取率分别提高41.0%和29.1%;与乙醇提取比较,酶法提取分别提高了30.9%和21.8%与酸水提取法、大孔吸附树脂提取法、半仿生提取法、乙醇提取法相比,纤维素酶催化转化得到的乌头碱和乌头总碱提取率显著增加,说明采用纤维素酶对川乌药材进行预处理,酶催化破坏了植物细胞壁上的β-D-葡萄糖苷键,使包裹生物碱的纤维素大量分解,进而破坏细胞壁,增加细胞壁的通透性,减小传质阻力,从而有效提高了生物碱成分的溶出。纤维素酶催化转化法对乌头生物碱的提取率的提高有明显的促进作用,且纤维素酶催化具有条件温和、作用时间短等优点,可在实际生产中推广应用。更多还原

【Abstract】 Owing to the deletion of petrochemical resources and the energy shortage, environment and society crisis were acceleratly deteriorating. As a promising strategy for developing green chemistry and green chemical engineering, biocatalysis technology will exert significant influence on medicine, agriculture, food, life science and so on. Biocatalysis is a new technology for extracting and separating active constituents from natural plants via enzyme catalysis. The plant cell walls usually hinder the extraction of effective constituents. If proper enzymes are employed, the cell walls will be decomposed, the extraction rate will be improved and the cost thus be reduced.Up to now, no studies of cellulose-assisted extraction of aconitum alkaloids have been reported both at home and broad. This preliminarily study was to explore the reaction condition and underlying mechanism of aconitum alkaloids catalyzed and transformed by cellulase.In this present study, the extraction rate of aconitine and aconitum alkaloids were regarded as indexes. The impact of aconitum alkaloids rate through enzyme-catalyzed reaction was studied by single factor experiment to determine the scope of the temperature, pH, cellulase dosage and reaction time. The optimum conditions of enzymatic extraction have been obtained by orthogonal test and compared with acid extraction, macroporous adsorbing resins extraction, semi-bionic extraction and alcohol extraction. Cellulase was recruited to pretreat the prepared Radix Aconiti, subsequently the aconitum alkaloids were extracted by alcohol. The optimum catalysis conditions of cellulase and its effect on the extraction rate of aconitum alkaloids were studied. The extraction rate and purity by cellulase catalysis synergism method was compared with others. Meanwhile, the effect of cellulase catalysis on conventional alcohol extraction and the mechanism of cellulase catalysis were also studied.The optimum conditions that cellulase catalyzed and transformed aconitum alkaloids were at 45℃, pH 4.5, with cellulase dosage of 0.8mg/g(0.01g cellulase in 1 g material) and 4 h reaction time of cellulase. The extraction rates of aconitine and aconitum alkaloids of semi-bionic extraction were 0.002 447%, 0.244 410% respectively. Compared with the acid extraction, the extraction rates of aconitine and aconitum alkaloids of cellulose enzyme extraction increased by 84.4%, 59.3% respectively. Compared with the macroporous adsorbing resins extraction, the extraction rates of aconitine and aconitum alkaloids increased by 61.1%, 41.4%, respectively. Compared with the semi-bionic extraction, the extraction rates of aconitine and aconitum alkaloids increased by 41.0%, 29.1%, respectively. Compared with the alcohol extraction, the extraction rates of aconitine and aconitum alkaloids increased by30.9%, 21.8%, respectively. Compared with acid extraction, macroporous adsorbing resins extraction, semi-bionic extraction, alcohol extraction, the cellulose enzyme extraction rates of aconitine and aconitum alkaloids were much higher. It indicated that the enzyme catalysis destroyβ-D-glucoside bond of the cell wall and decomposed cellulose and minished mass transfer resistance. It made aconitum alkaloids be taken out easier. The yield rate of aconitum alkaloids improved obviously after the prepared radix aconiti was pretreated by cellulase. Cellulase catalysis method significantly improve the extration rate of aconitum alkaloids, which has advantages of mild conditions, short time and so on. This study suggested that the cellulase catalysis technology is promising in extracting active ingredients from natural plants and also feasible in practical production.更多还原