【作者】 孙崇德；

【导师】 张上隆； 陈昆松；

【作者基本信息】 浙江大学 ， 果树学， 2006， 博士

【摘要】 以胡柚（C．changshanensis）、柚（C．grandis）、温州蜜柑（C．unshiu）和椪柑（C．reticulata）的果实、根、枝以及叶片等不同器官和组织为试验材料，改进并建立了柠檬苦素类化合物和吖啶酮生物碱的检测与提取方法，研究了不同柑桔的不同发育阶段、不同器官和组织中柠檬苦素和诺米林的含量变化，检测了不同柑桔果实和根粗提物的抗氧化活性及柚果实囊衣柠檬苦素和诺米林粗结晶的抗氧化和抗菌活性。主要结果如下：建立了一个简单、方便、高效的柑桔柠檬苦素和诺米林检测提取方法。TLC与HPLC两种方法相结合可有效地对柑桔柠檬苦素和诺米林进行定性和定量检测。HPLC的检测体系为：检测波长210nm，色谱柱为C₁₈柱，甲醇:乙腈:磷酸缓冲液（pH=3.5）=10:40:39作为流动相；柠檬苦素和诺米林的RSD（n=5）分别为6.48和2.90％，检测限（S／N=3）均为0.03μg／g。柑桔柠檬苦素和诺米林的提取采用二氯甲烷为提取液，50℃水浴提取60min，柠檬苦素和诺米林的回收率分别可达90.45和91.09％。根据9（10H）吖啶酮结构荧光特性建立的HPLC检测条件为：激发波长和发射波长分别为395和435nm，色谱柱为C₁₈柱，以去离子水（A）和甲醇（B）为流动相采用梯度洗脱的方法。该检测体系操作简单，灵敏度高，稳定性好，得到的检测限（S／N=3）为0.67ng／g，在0.25-4.00μg／mL浓度范围内RSD（n=5）为1.29-2.85％。柑桔9（10H）吖啶酮提取的优化体系采用乙醇为提取液，在频率和功率分别为26kHz和500W的条件下超声提取60min（30℃），得到9（10H）吖啶酮的回收率在97.12-101.56％范围内。在果实发育过程中，不同柑桔果实的油胞层、白皮层、囊衣和汁胞四种组织中柠檬苦素和诺米林的含量变化总体都呈现先上升后下降的趋势；柠檬苦素在椪柑油胞层和胡柚囊衣中的最高含量分别达到1.53和3.52mg／g，诺米林在柚油胞层和胡柚囊衣中的最高含量分别达到2.82和2.92mg／g；柚白皮层中的柠檬苦素和诺米林的含量在发育过程中保持较低的水平，始终分别低于0.09和0.08mg／g；在成熟柚果实的囊衣中，柠檬苦素和诺米林含量呈现回升的现象，含量由采前3w的0.0001和0.06mg／g分别回升到了采收时的0.42和0.51mg／g。对于柑桔不同器官的研究表明，柠檬苦素和诺米林在柑桔的根中大量积累，二者总含量高于同期的果实、叶片和枝中的含量，在椪柑的须根中柠檬苦素和诺米林的总含量达到2.56mg／g，同期果实、叶片和枝的含量分别为1.35、0.64和0.36mg／g。柚果实囊衣提取的柠檬苦素和诺米林粗品的抗氧化活性研究结果表明，粗品的抗氧化活性是等量Vc的10.34倍，四个品种柑桔根中柠檬苦素和诺米林粗提物的抗氧化活性是等量Vc的12.59-17.21倍。柚果实囊衣提取的柠檬苦素和诺米林粗结晶对细菌和霉菌生长有不同程度的抑制效应。该粗结晶对青霉和黑曲霉的抑菌圈直径分别为2.09和2.25cm（设定的对照分别为1.26和1.25cm），但对细菌抑制效应不明显，抑菌圈直径分别为1.27和1.28cm（设定的对照分别为1.27和1.26cm）。柑桔柠檬苦素和诺米林的含量和生物活性变化均呈现遗传、发育阶段、器官和组织的多样性。简便、稳定的柑桔9（10H）吖啶酮检测与提取体系建立，为进一步开展柑桔吖啶酮生物碱的含量和生物活性变化研究提供了一个重要的技术支持。更多还原

【Abstract】 In the present study, we modified and established the method for determination and extraction of limonin, nomilin and acridone in different organs （fruit, root, stem and leaf） and tissues of C. changshanensis, C. grandis, C. unshiu, and C. reticulate. In addition, the contents and bioactivities such as antioxidant and antibacterial activity were evaluated for limonin and nomilin from citrus fruit or root extracts of different cultivars, developmental stages, tissues and organs, and those in the raw crystal of limonin and nomilin from segment membrane （SM） of C. grandis. The main results were as follows:The developed method for determination and extraction of limonin and nomilin from citrus were simple, easy-to-do, and high efficient. Both TLC and HPLC were combined for the quantification and qualification of limonin and nomilin from different citrus samples. The HPLC conditions were: detection wavelength were 210 nm; C₁₈ column, methanol: acetonitrile: phosphate buffer （pH=3.5） =10:40:39 were used as the mobile phase; the relative standard deviation （RSD） for limonin and nomilin were 6.48 and 2.90% （n=5）, respectively; the limit of detection （LOD） for both compounds were 0.03μg/g （S/N=3）. For extraction of limonin and nomilin from citrus, methylene chloride were used as the extraction solution, after 60 min of water bath extraction at 50℃, the recoveries for limonin and nomilin were 90.45 and 91.09%, respectively.The HPLC detection system, which were established based on the fluorescent characteristics of 9（10H）acridone structure, included: an excitation wavelength of 395 nm and an emission wavelength of 435 nm, a gradient combination of deionized water （A） and methanol （B） were used as the elute solution. Such detection system were proved to be high sensitivity and stability, and the limit of detection （LOD） were 0.67 ng/g （S/N=3）, the relative standard deviation （RSD） for the contents between 0.25-4.00μg/mL were 1.29-2.85% （n=5）. The developed method for extraction and determination of acridone used ethanol as the extraction solution, after 60 min ultrasonic extraction at a frequency of 26 Hz and a power of 500 W at 30℃, the recovery of 9 （10H） acridone were between 97.12-101.56%.During the fruit development, there was an increase followed by a decrease for the contents of limonin and nomilin in the citrus fruit of all the four tissues studied, i.e. flavedo, albedo, SM, and juice vesicle （JV）. Highest limonin contents were found in the flavedo of C. reticulat and segment membrane （SM） of C. changshanensis, which were 1.53 and 3.52 mg/g, respectively. Highest nomilin contents were found in the flavedo of C. grandis and SM of C. changshanensis, which were 2.82 and 2.92 mg/g, respectively. Both the limonin and nomilin cotents in the albedo of C. Grandis kept at low level throughout the developmental stages, which were lower than 0.09 and 0.08 mg/g, respectively. In the SM of mature C. Grandis fruit, both the limonin and nomilin increased from the 0.0001 and 0.06 mg/g at 3w before harvest to 0.42 and 0.51 mg/g at harvest. The results of study of different citrus organs showed an aboundant accumulation of both the limonin and nomilin in the ctirus root, the total amount of which were higher than their counterparts in the fruit, leaf and stem. The total amount of limonin and nomilin contents in the fibre of C. reticulate were 2.56 mg/g, while the corresponding amount in the fruit, leaf and stem of the same developmental stage were 1.35, 0.64, and 0.36 mg/g, respectively.Study of antioxidant activity of the raw crystal of limonin and nomilin from SM of C. grandis showed: the antioxidant capacity of limonoid raw crystal of citrus fruit was 10.34 times that of Vc; the antioxidant capacity of crude extract of citrus root was 12.59-17.21 times that of Vc. Different inhibition effect were found for the limonin, nomilin and acridone extract from citrus fruit or root against the growth of two bacteria and two fungus. The diameter of inhibition circle of limonin and nomilin extract against Penicillium digitatum and Aspergillus niger were 2.09 and 2.25 cm, respectively （the control were 1.26 and 1.25 cm, respectively）, however, it showed no effect on the growth of Escherichia coil and Staphylococcus aureus, the diameter of inhibition circle were 1.27 and 1.28 cm, respectively （the control were 1.27 and 1.26 cm, respectively）.The present study showed a genetic, developmental and tissue diversity of both the content and bioactivity of limonin and nomilin in citrus. The developed detection and extraction system for 9 （10H） acridone were easy-to-do and stable, which could be used for further study of content and bioactivity of acridone alkaloid in citrus.更多还原