【作者】 刘亮；

【导师】 戚向阳；

【作者基本信息】 华中农业大学 ， 食品科学， 2006， 硕士

【摘要】 枳实为芸香科植物酸橙及其栽培变种或甜橙的干燥幼果及未成熟果实。目前对其有效成分的研究主要集中在生物碱、黄酮和挥发油等方面，而对枳实中柠檬苦素类似物的研究鲜有报道。近年来，柠檬苦素类似物一直是国外研究的热点，大量研究表明柑桔及其副产物富含柠檬苦素类似物，并且该类物质具有抗癌、昆虫拒食和抗氧化等活性。枳实作为柑桔的一种，含有一定量的柠檬苦素。因此，本文以枳实加工副产物为原料，对其中柠檬苦素类似物的提取、分离纯化、分析鉴定、抗氧化及抗菌活性进行了系列研究，主要研究结果如下：1柠檬苦素类似物甙元的分析建立了柠檬苦素的高效液相色谱分析方法。色谱条件为：色谱柱：PhenomenexC₁₈柱（250×4.6mm，5μm）；流动相：乙腈／水=45／55（v／v）；流速：1mL／mim；柱温：25℃；检测波长：210nm。经考察该方法对柠檬苦素和诺米林均有良好的线性关系，相关系数分别为0.9986和0.9992，加标回收率分别99.82％～102.99％和92.80％～100.63％，精密度分别为2.55％和2.30％，且该方法简便、快速，可用于柑桔及其副产物中柠檬苦素和诺米林的定量分析。用建立的高效液相色谱分析方法对枳实加工副产物和不同柑桔品种中的柠檬苦素和诺米林的含量进行了分析，结果表明：柠檬苦素和诺米林两种主要甙元的含量因柑桔品种、组织及成熟度而异。枳实加工副产物中柠檬苦素的含量高于其它柑桔品种。就不同柑桔品种而言，柑桔种子和外皮中甙元的含量要高于内皮、瓣膜中的含量，果肉中含量最低。半成熟品种中两种甙元的含量高于成熟品种。外皮中，诺米林的含量高于柠檬苦素的含量，而种子中柠檬苦素的含量要高于诺米林。2柠檬苦素提取条件的研究通过单因素实验和旋转正交实验确定了枳实加工副产物中柠檬苦素的最佳提取条件为：6倍于原料的78％乙醇（pH 6.5）于60℃下回流提取1次，提取时间为2h。且该方法成本低，无毒性溶剂残留，有利于工业化生产。3柠檬苦素类似物的分离纯化用大孔树脂和葡聚糖凝胶柱层析分离纯化柠檬苦素类似物。大孔树脂的最佳柱分离条件为：将上样液的pH调为7.5，上柱，依次用pH7.5的水、50％乙醇、70％乙醇进行洗脱，收集70％乙醇洗脱液，经浓缩，冷冻干燥可得柠檬苦素类似物粗提物（FAC）。该工艺制备产品的得率为3.21％，柠檬苦素的含量为4.55％。将大孔树脂分离得到的粗提物FAC用少量乙醇溶解后用Sephadex LH-20凝胶柱进一步纯化，其最佳分离条件：将上样后的样品依次用50％乙醇、70％乙醇进行洗脱，每梯度收集40管，流速为0.5mL／min，12min／管。各管用Ehrich’s试剂显色后在400nm处比色，根据吸光值的大小绘制色谱图，共得到五个色谱峰，分别收集各峰，浓缩、冷冻干燥可得五级分（分别为FA1、FA2、FA3、FA4、FA5）。经高效液相色谱分析和紫外光谱初步鉴定表明，只有FA4中含有柠檬苦素，其含量为9.96％。4柠檬苦素类似物糖甙的分析及鉴定根据文献报道，建立了柠檬苦素类似物糖甙的高效液相色谱分析方法。色谱条件为：色谱柱：Phenomenex C₁₈柱（250×4.6mm，5μm）；流动相：乙腈（A）-3mM磷酸（B）二元梯度洗脱，15％A（Omin）～26％A（40min）；检测波长：210nm；流速：1mL／min；柱温：25℃。高效液相色谱及紫外光谱分析表明：FA2、FA3、FA4、FA5中不含柠檬苦素类似物糖甙，FA1中可能存在糖甙成分，其HPLC-ESI／MS／MS的分析结果表明：FA1中存在的糖甙成分可能为诺米林酸甙。5不同枳实提取物的生物活性通过体外实验研究了不同提取物清除羟基自由基的能力和抑菌活性。清除羟基自由基的实验结果表明：FA1～FA4在400μg／mL～2000μg／mL对·OH自由基有不同程度的清除作用，以FA3的清除效果最好。在200μg／mL时，柠檬苦素和诺米林的抗氧化活性高于FA1和FA4，而低于FA3。这可能与FA3中含有大量黄酮类物质和FA1、FA4所含柠檬苦素类似物有关。抑菌实验结果表明：各级分对细菌都有不同程度的抑制作用，而对霉菌和酵母菌几乎无抑制作用。其中FA1和FA3对细菌的抑制效果较好，FA4次之。温度和pH值对抑菌活性有较大影响，低温处理和微酸性条件有利于抑菌活性的发挥。但各级分中的有效成分还有待于进一步的深入研究。更多还原

【Abstract】 Fructus Aurantii Immaturus is dry immature fruit of Citrus aurantium L. and its variation or Citrus sinensis Osbenkr of of Rutaceae. Presently, researches on bioactive components of Fructus Aurantii Immaturus are focus on alkaloids, flavonoids, volatile oil, and so on, but there are few reports on limonoids from Fructus Aurantii Immaturus. Now, the studies on limonoids are very popular abroad. Citrus and Citrus by-products are rich in limonoids which have anticarcinogenic activity, antifeeding activity, antioxidant activity and so on. Fructus Aurantii Immaturus also contain limonoids. So the extraction, isolation and purification, analysis and determination of limonoids from by-products of Fructus Aurantii Immaturus were studied in this paper, and antioxidant effect and antibacteria activity of different extracts were also discussed. These main results were indicated as follows:1 Analysis of limonoid aglycosidesHPLC analysis method of limonin and nomilin was established. The samples analysis were performed on C₁₈ Phenomenex, 5μm（250×4.6mm）column, and a binary mobile phase consisting of acetonitrile/water （45/55）was used with a flow rate of1.0mL/min at 25℃, the elution was monitored by UV absorption at 210nm.The methodhad good linear relationship（R²=0.9986 and R²=0.9992）. Recovery ratios were 99.82%～102.99% and 92.80%～100.63%, and relative standard deviations were 2.55% and 2.30% respectively. So the method was simple, accurate and rapid, and was available in analysis of limonin and nomilin from Citrus or its by-products.Limonin and nomilin from by-products of Fructus Aurantii Immaturus and different Citrus were analyzed. The results showed that the content of limonin and nomilin varied with variety, tissue and maturity of Citrus. The limonin content was higher in by-products of Fructus Aurantii Immaturus than that in the others. For different Citrus, the content of aglycosides was higher in seeds and flavedo than that in segment membrane and albedo, and was the least in juice vesicle. Besides, the aglycosides content was higher in the immaturity than that in the maturity, and nomilin content was much more than limonin content in flavedo, but was the reverse in flavedo. 2 Reseaches on extract conditions of limoninThe optimum extract condition of limonin from by-products of Fructus Aurantii Immaturus was studied by means of single factor experiment and rotational orthogonal experiment. The result indicated that the extracting efficiency was best when the sample was extracted with pH 6.5, 78% ethanol solution （material/solvent=1/6, w/v） at 60℃and for 2h. The mothod was cost-effective and suitable to industrialization.3 Isolation and purification of limonoidsThe isolation and purification of limonoids from Fructus Aurantii Immaturus by column chromatography with macroporous resin and Sephadex LH-20 was investigated. The sample liquor was transferred to the top of column packed with macroporous resin after adjusting pH value 7.5, then washed thoroughly with water at pH 7.5, 50% ethanol, 70% ethanol successively, the 70% ethanol eluate was collected, concentrated and freeze-dried. And the limonoids crude extract from Fructus Aurantii Immaturus （FAC） was obtained with the limonin content of 4.55% and products yield 3.21%.FAC was further purified by Sephadex LH-20. FAC was continuously eluted with 50% and 70% ethanol with the flow rate 0.5mL/min, 6mL per tube, the column was eluted with 240mL of each solvent mixture. After displaying colour with Ehrich’s reagent, the absorption value was determined at 400nm. Then the chromatograpy was portrayed in accordance with absorbence absorption value and five chromatography peaks were abtained. The elution was collected according to chromatogrphy, then, concentrated, freeze-dried repectively and five extracts were abtained （FA1, FA2, FA3, FA4, FA5）. Each extract was indentified by HPLC and UV-spectrum and the result showed that limonin was only in FA4 and its limonin content was 9.96%.4 Analysis and identification of limnoid glucosideHPLC method of analysis limonoid glucoside was developed. C₁₈ reverse-phase, Phenomenex, 5μm （250×4.6mm） column was used, and the column being eluted at 1mL/min with a linear gradient system, starting with 15%acetonitrile in 3mM H₃PO₄ and ending with 26% after 40min. The elution was monitored by UV absorption at 210nm. Each extract was analysed by HPLC and UV spectroscopy. The results showed that FA2、FA3、FA4、FA5 didn’t contain limonoid glucoside, except that FA1. According to HPLC-ESI/MS/MS analysis, the main limonoid glucoside in FA1 might be nomilic acid glucoside.5 Bioactivities of different Fructus Aurantii Immaturus extractsThe antioxidant effects and antibacteria activity of different extracts in vitro were studied. FA1～FA4 had scavenging effects on hydroxyl radical （·OH） from 400μg/mL to 2000μg/mL, and FA3 had the best scavenging effect among the extracts. The antioxidant effects of limonin and nomilin were higher than that of FA1, FA4 at 200μg/mL, but lower than FA3. That may be related to antioxidant effect of the flavonoids and limonoids.Each extract had inhibiting effects on bacteria, and little effect on mould and yeast. Antibacterial effects of FA1 and FA4 were better than others. Temperature and pH value had an influence on inhibiting effects of each extract. Low temperature treatment and mildly acidic condition were favorable to antibacterial effects. But the bioactive components of each extract need to be further studied.更多还原