枳实加工副产物中柠檬苦素类似物的分离、分析及活性研究
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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：PhenomenexC_(18)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 C_(18)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。
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 C_(18) 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(R~2=0.9986 and R~2=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 limonin
The 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 limonoids
The 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 glucoside
HPLC method of analysis limonoid glucoside was developed. C_(18) 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 H_3PO_4 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 extracts
The 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.
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：PhenomenexC_(18)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 C_(18)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。
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 C_(18) 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(R~2=0.9986 and R~2=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 limonin
The 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 limonoids
The 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 glucoside
HPLC method of analysis limonoid glucoside was developed. C_(18) 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 H_3PO_4 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 extracts
The 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.
引文
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15．田庆国．甜橙种子中柠檬苦素类化合物的提取．林产化学与工业，1999，(9)：71-74
16．田庆国，丁霄霖．测定橘核中柠檬苦素类似物的分光光度法．分析测试学报， 1999，18(5)：45-47
17．田庆国．无锡轻工大学博士论文．1999
18．汪海峰，朱军莉，刘建新．新型饲料添加剂．柠檬苦素类似物．广东饲料，2003，12(5)：31-32
19．周萍．中国民间百草良方．湖南科学技术出版社．2002，475
20．张哲，孙健，温庆辉．柠檬苦素及其类似物的抗肿瘤活性研究进展．吉林中医药，2005，25(5)：61-62
21．张虹，张艳萍．柠檬苦素类似物的研究进展．食品与发酵工业，2002，28(2)：80-83
22．朱玲，杨峰，唐德才等．枳实的药理研究进展．中医药学报，2004，32(2)：64-66
23. Arigoni D, Barton D H R, Corey E J. The constitution of limonin. J. Experientia, 1960, 16:41-49
24. Ayano S, Miyake M, Ifuku Y, Ishikawa H. Extraction of Limonoids using micro bubble supercritical carbon dioxide. Nippon Shokuhan Kogyo Gakkaishi, 1996, 43: 395
25. Abbasi S, Zandi P, Mirbagheri E. Quantitation of limonin in Iranian orange juice concentrates using high-performance liquid chromatography and spectrophoto-metric methods. European Food Research and Technology, 2005, 211(1-2): 202-207
26. Bernay S. Limonin. Annalen, 1841, 40:317-319
27. Barton D H R, Prandhan S K, Sternhell S. Triterpenoids. Part ⅩⅩⅤ. The constitution of limonin and related bitter principles. J. Am. Chem. Soc., 1961, 255-275
28. Bennett R D, Hasegawa S. 7α-Oxygenated limonoids from the Rutaceae. Phytochemistry, 1982, 21(9): 2349-2354
29. Barry H, John M, Okezie I. The deoxyribose method: A simple "Test-Tube" assay for determination of rate constants for reactions of hydroxyl radicals. Analytical Biochemistry, 1987, 165:215-219
30. Bennett R D, Hasegawa S, Herman Z. Glucosides of acidic limonoids in citrus. Phytochemistry, 1989, 28(10): 2777-2781
31. Braddock R J, Bryan C R. Extraction parameters and capillary electrophoresis anal ysis of limonin glucoside and phlorin in citrus by-products, J. Agric. Food Chem. , 2001, 49:5982-5988
32. Battinelli L, Mengoni F, Lichtner M. Effect of limonin and nomilin on HIV-1 replication on infected human mononuclear cells. Planta Med., 2003, 69:910-913
33. Dreyer D L. Citrus bitter principles-Ⅱ. application of NMR to structure and steroch-emical problems. Tetrahedron, 1965, 21: 75-87
34. Dekker R H. De-bittering of citrus fruit juices: Specific removal of limonin and other bitter principles. Aust. J. Biotech. , 1988, 2:65-76
35．Douglas C．Montgomery著，汪仁官，陈荣昭译．实验设计与分析．北京：中国统计出版社，1998，589
36. Emerson O H. The bitter principles of citrus fruit. Ⅰ. Isolation of nomilin, a new bitter principle from the seeds of oranges and lemons. Am. Chem. Soc., 1948, 70(1): 545-549
37. Emerson O H. Bitter principles of citrus. Ⅱ. Relation of nomolin and obacunone. Am. Chem. Soc., 1951, 76(3): 2621-2623
38. Fong C H, Hasegawa S, Charles W, Coggins Jr, Darwin R Atkin, Miyake M. Contents of Limonoids and Limonin 17-β-D-Glucopyranoside in Fruit Tissue of Valencia Orange during Fruit Growth and Maturation. J. Agric. Food Chem., 1992, 40: 1178-1181
39. Fong C H, Hasegawa S, Miyake M. Limonoids and their glucosides in Valencia orange seeds during fruit growth and development, J. Agric. Food Chem., 1993, 41:112-115
40. Govindachari T R., Suresh G, Gopalakrishnan G. Antifungal activity of some tetranortriterpenoids. Fitoterapia, 2000, 71: 317-320
41. Hasegawa S. Limonoids in Citrus seeds: Origin and relative concentration. J. Agric. Food Chem., 1980, 28:922-925
42. Hasegawa S, Bennett R D, . Limonoid glucosides in Citrus. Phytochemistry, 1989, 28(6): 1717-1720
43. Herman Z, Fong C H, Ou P, Hasegawa S. Limonoid glucosides in orange juices by HPLC. J. Agric. Food Chem., 1990, 38:1860-1861
44. Hasegawa S, Ou P, Fong C H, Herman Z, Charles W, Coggins Jr, Darwin R A. Change in the limonoate A-ring lactone and limonin17-β-D-glucopyranoside content of navel oranges during fruit growth and matruation. J. Agric. Food Chem., 1991, 39:262-265
45. Hasegawa S, Berhow M A. Analysis of limonoids by thin-layer chromatography. In: Berhow M A. Hasegawa S. Manners G D eds., Citrus limonoids: Functional chemicals in agriculture and foods. American chemical society, Washington, DC., 2000. 31-59
46. Haswgawa S. Biochemistry of limonoids in Citrus. In: Berhow M A. Hasegawa S. Manners G D eds., Citrus Limonoids: Functional Chemicals in Agriculture and Foods. American Chemical Society, Washington, DC., 2000. 21
47. Jack D. Citrus compound-ready to help your body. Agricultural research, 2005, 2: 16-17
48. Jitpukdeebodintra S, Chantachum S, Ratanaphan A. Stability oflimonin from lime seeds. Electron. Environ. Agric. Food Chem., 2005, 4(3): 217-223
49. Kelly C, Jewell C, O'Brien N M. The effect of dietary supplementation with the citrus limonoids, limonin and nomilin on xenobiotic-metabolizing enzymes in the liver and small intestine of the rat. Nutrition Research, 2003, 23:681-690
50. Lam L K T, Li Y, Hasegawa S. Effect of citrus limonoids on glutathione s-transferase activity in mice. J. Agric. Food Chem., 1989,37:878-880
51. Liminoids strongly inhibit HIV protease activity[N]. Health & Medicine Week, 2004, 2/9
52. Maier V P, Brewster L C, Hsu A C. Specific thin-layer chromatography assay of limonin. J. Agric. Food Chem. , 1973, 18:250-252
53. Maier V P, Bennett R D, Hasegawa S. Limonin and other limonoids. In: Maier V P ed., Citrus Science and technology. INC., A Ⅵ Publishing company, 1977, 1: 355-396
54. Miller E G, Gonzales-Sanders A P, Couvillon A M. Inhibition of hamster buccal pouch carcinogenesis by limonin .17-β-D-glucopyranoside. Nutrition Cancer. 1992, 17(1): 1-7
55. Mendel M J, Alford A R, Rajab M S. Relationship of citrus limonoid structure to feeding deterrence against fall armyworm (Lepidoptera: Noetuidae) larvae. Environmental Entomology, 1993, 22(1): 167-173
56. Moodley V E, Mulholland D A, Raynor M W. Mieellar eleetrokinetie capillary chromatography of limonoid glucosides from citrus seeds. J. Chromatogr. A, 1995, 718:187-193
57. MeIntosh C A, Mansell R L. Three-dimensional distribution of limonin, limonoate A-ring monolaetone, and narigin in the fruit tissues of three varieties of Citrus paradise. J. Agric. Food Chem., 1997, 45:2876-2883
58. Matsuda H, Yoshikawa M, Iinuma M, Kubo M. Antinociceptive and antiinflammatory activites of limonin isolated from the fruits of Evodia rutaecarpa Var. bodinied. Planta Med., 1998, 64(4): 339-342
59. Manners G D. Hasegawa S. A new normal phase liquid chromatographic method for the analysis of limonoids in Citrus. Phytochem. Anal., 1999, 10:76-81
60. Murray K D, Hasegawa S, Alford A R. Antifeedant activity of citrus limonoids against Colorado potato beetle: Comparison of aglycones and glucosides. Entomologia Experimentalis Applicata, 1999, 92(3): 331-334
61. Miyake M, Shimoda M, Osajima Y, Inaba N, Ayano S, Ozaki Y, Hasegawa S. Extraction and recovery of limonoids with the supercdtical carbon dioxide micro-bubble method. A CS Symp Ser., 2000, 758: 96-106
62. Moriguchi T, Kita M, Hasegawa S, Omura M. Molecular approach to citrus flavonoid and limonoid biosynthesis. J. Food, Agriculture & Environment. 2003, 1(1): 22-25
63. Manners G D, Breksa Ⅲ A P. Identifying citrus limonoid aglycones by HPLC-EI/MS and HPLC-APCI/MS. Phytochemical Analysis, 2004, 15(6): 372-381
64. Nawwar M A M, Marzouk M S, Nigge W. High-performance liquid chromatographic/Electrospray ionization mass spectrometric screening for polyhenolic compounds of Epilobium hirostum-the structure of the unique ellagitannin epilobamide-A. Mass Spectrometry, 1997, 32: 645-654
65. Ozaki Y, Fong C H, Herman Z, Maeda H, Miyake M, Ieuku Y, Hasegawa S. Limonoid glucosides in Citrus seeds. Biol. Chem., 1991, 55(1): 137-141
66. Ohta H, Chi H Fong, Berhow M, Hasegawa S. Thin-layer and high-performance liquid chromatographic analyses of limonoids and limonoid glucosides in Citrus seeds. J. Chromatogr., 1993, 639:295-302
67. Ozaki Y, Ayano S, Inaba N, Miyake M, Bernow Mark A, Hasegawa S. Limonoid glucosides in fruit, juice and processing by-products of Satsuma Mandarin (Citrus unshiu Marcov.). Food Sci., 1995, 60(1): 186-189
68. Poulose SM, Harris E D. Citrus limonoids induce apoptosis in human neuroblastoma cells and have radical scavenging activity. Nutrition, 2005, 135(4): 870-877
69. Rouseff R L, Fisher J F. Determination of limonin and related limonoids in citrus juices by high performance liquid chromatography. Anal. Chem., 1980, 521: 228-1233
70. Rorseff R L, NaGY S. Distribution of limonoids in Citrus seeds. Phytochemistry, 1982, 21:85-90
71. Raman G, Cho M, Brodbelt J S. Isolation and purification of closely related Citrus limonoid glucosides by flash chromatography. Phytochemical Analysis, 2005, 16(3): 155-160
72. Shaw P E, Wilson C W. A rapid method for the determination of limonin in citrus juices by high performance liquid chromatography. J. Food Sci. , 1984, 49: 1216-1218
73. Shaw P E. The use of a simple solvent optimization program to improve separation of limonin in citrus juices. J. Chromatogr. Sci., 1986, 24:364-366
74. Sawabe A, Morita M, Kiso T, Kishine H, Ohtsubo Y, Minematsu T, Matsubara Y, Okamoto T. Isolation and characterization of new limonoid glycosides from Citrus unshiu peels. Carbohydrate Research, 1999, 315:142-147
75. Schoch T, Manners G H, Hasegawa S. Analysis of limonoid glucosides from Citrus by electrospray ionization liquid chromatography-mass spectrometry. J. Agric. Food Chem. , 2001, 49(3):-1102-1108
76. Saipctch K S. Separation and quantitation of limonoids and flavonoids in juice and by-products of sweet orange (Citrus sinensis). (Ph D dissertation). Michigan State University, 2004
77. Sun C D, Chcn KS, Chert Y, Chen Q J. Contents and antioxidant capacity of limonin and nomilin in different tissues of citrus fruit of four cultivars during fruit growth and maturation. Food Chemistry, 2005,93(4): 599-605
78. Tatum J H, Berry R E. Method for estimating limonin content of citrus juices. Food Sci. ,1973,38:1244-1246
79. Tian Q G, Ding X L. Screening for limonoid glucosides in Citrus tangerine (Tanaka) Tseng by high-performance liquid chromatography-electrospray ionization mass spectrometry. J. Chromatography A, 2000, 847: 13-19
80. Tian Q G, Edward G, Miller, Ahmad H. Differential Inhibition of Human Cancer Cell Proliferation by Citrus Limonoids. Nutrition and cancer, 2001, 40(2): 180-184
81. Tian Q G, Schwartz S J. Mass spectrometry and tandem mass spectrometry of Citrus Limonoids. Anal. Chem. ,2003,75:5451-5460
82. Tian Q G, Kent K D, Bomser J A, Schwartz S J. Characterization of limonin glucoside metabolites from human prostate cell culture medium using high-performance liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry. Rapid Commun. Mass Spectrum, 2004, 18: 3099-3104
83. Van Beek T A, Blaakmeer A. Determination of limonin in grapefruit juice and other citrus juices by high performance liquid chromatography. J. Chromatogr., 1989, 464: 375-386
84. Weiler E W, Mansell R E. Radioimmunoassay of limonin using a tritiated tracer. J. Agric. Food Chem. ,1980,28:543-545
85. Widmer W W. Improvements in the quantitation of limonin in citrus juice by reversed phase high-performance liquid chromatography. J. Agric. Food Chem. , 1991,39: 1472-1476
86. Wada K, Yagi M, Kurihara T, Haga M. Studies on the constituents of edible and medicinal plants. III. Effects of seven limonoids on sleeping time induced in mice by anesthetics. Chem Pharm Bull, 1992,40(11): 3079-3080
87. Yu J, Wang L, Walzem R L, Miller E D, Pike L M, Patil B S. Antioxidant activity of citrus limonoids, flavonoids, and coumarins. J. Agric. Food Chem. , 2005, 53: 2009-2014
2．范秀容，李广武，沈萍．微生物学实验(第二版)[M]．北京：高等教育出版社，1989
3．国家药典委员会，中华人民共和国药典(2000年版一部)．北京：化学工业出版社，2000，200-201
4．湖南医药工业研究所四室．枳实升压成分的化学研究简报．中草药通讯，1976，5：198
5．胡盛珊，王大元，邱萍，俞红．枳实有效成分的药理活性比较．中草药，1994，25(8)：419-421
6．廖凤霞，孙冠芸，杨致邦，夏之宁．枳实挥发油的化学成分分析及其抗菌活性的研究．中草药，2004，35(1)：20-22
7．李福兴，王建江．柠檬苦素化合物的研究现状．国外医学(中医中药分册)，2000，22(3)：131-136
8．赖作企，符雄，沙田柚种子化学成分的研究．中山大学学报，1995，4：117-121
9．刘中秋，蔡雄，赖小平，刘良．大孔树脂富集、纯化三七皂苷研究．中国实验方剂学杂志，2001，7(3)：47
10．李伯庭，王湘．大孔吸附树脂在天然产物分离中的应用．中草药，1992，14(4)：2-4
11．马秀敏，周晓英，张立，王亚南，丁剑冰，田树革等．天山堇菜提取物的体外抗菌实验．时珍国医国药，2004，15(8)：470-471
12．彭国平，牛贺明，徐丽华．枳实活性成分研究．南京中医药大学学报，2001，17(2)：91-92
13．戚向阳，王小红，陈维军，王清章，彭光华．不同苹果多酚提取物对油脂抗氧化效果的研究．中国油脂，2001，26(4)：51-53
14．沈保安．中国常用中草药．合肥：安徽科学技术出版社．2005，182-184
15．田庆国．甜橙种子中柠檬苦素类化合物的提取．林产化学与工业，1999，(9)：71-74
16．田庆国，丁霄霖．测定橘核中柠檬苦素类似物的分光光度法．分析测试学报， 1999，18(5)：45-47
17．田庆国．无锡轻工大学博士论文．1999
18．汪海峰，朱军莉，刘建新．新型饲料添加剂．柠檬苦素类似物．广东饲料，2003，12(5)：31-32
19．周萍．中国民间百草良方．湖南科学技术出版社．2002，475
20．张哲，孙健，温庆辉．柠檬苦素及其类似物的抗肿瘤活性研究进展．吉林中医药，2005，25(5)：61-62
21．张虹，张艳萍．柠檬苦素类似物的研究进展．食品与发酵工业，2002，28(2)：80-83
22．朱玲，杨峰，唐德才等．枳实的药理研究进展．中医药学报，2004，32(2)：64-66
23. Arigoni D, Barton D H R, Corey E J. The constitution of limonin. J. Experientia, 1960, 16:41-49
24. Ayano S, Miyake M, Ifuku Y, Ishikawa H. Extraction of Limonoids using micro bubble supercritical carbon dioxide. Nippon Shokuhan Kogyo Gakkaishi, 1996, 43: 395
25. Abbasi S, Zandi P, Mirbagheri E. Quantitation of limonin in Iranian orange juice concentrates using high-performance liquid chromatography and spectrophoto-metric methods. European Food Research and Technology, 2005, 211(1-2): 202-207
26. Bernay S. Limonin. Annalen, 1841, 40:317-319
27. Barton D H R, Prandhan S K, Sternhell S. Triterpenoids. Part ⅩⅩⅤ. The constitution of limonin and related bitter principles. J. Am. Chem. Soc., 1961, 255-275
28. Bennett R D, Hasegawa S. 7α-Oxygenated limonoids from the Rutaceae. Phytochemistry, 1982, 21(9): 2349-2354
29. Barry H, John M, Okezie I. The deoxyribose method: A simple "Test-Tube" assay for determination of rate constants for reactions of hydroxyl radicals. Analytical Biochemistry, 1987, 165:215-219
30. Bennett R D, Hasegawa S, Herman Z. Glucosides of acidic limonoids in citrus. Phytochemistry, 1989, 28(10): 2777-2781
31. Braddock R J, Bryan C R. Extraction parameters and capillary electrophoresis anal ysis of limonin glucoside and phlorin in citrus by-products, J. Agric. Food Chem. , 2001, 49:5982-5988
32. Battinelli L, Mengoni F, Lichtner M. Effect of limonin and nomilin on HIV-1 replication on infected human mononuclear cells. Planta Med., 2003, 69:910-913
33. Dreyer D L. Citrus bitter principles-Ⅱ. application of NMR to structure and steroch-emical problems. Tetrahedron, 1965, 21: 75-87
34. Dekker R H. De-bittering of citrus fruit juices: Specific removal of limonin and other bitter principles. Aust. J. Biotech. , 1988, 2:65-76
35．Douglas C．Montgomery著，汪仁官，陈荣昭译．实验设计与分析．北京：中国统计出版社，1998，589
36. Emerson O H. The bitter principles of citrus fruit. Ⅰ. Isolation of nomilin, a new bitter principle from the seeds of oranges and lemons. Am. Chem. Soc., 1948, 70(1): 545-549
37. Emerson O H. Bitter principles of citrus. Ⅱ. Relation of nomolin and obacunone. Am. Chem. Soc., 1951, 76(3): 2621-2623
38. Fong C H, Hasegawa S, Charles W, Coggins Jr, Darwin R Atkin, Miyake M. Contents of Limonoids and Limonin 17-β-D-Glucopyranoside in Fruit Tissue of Valencia Orange during Fruit Growth and Maturation. J. Agric. Food Chem., 1992, 40: 1178-1181
39. Fong C H, Hasegawa S, Miyake M. Limonoids and their glucosides in Valencia orange seeds during fruit growth and development, J. Agric. Food Chem., 1993, 41:112-115
40. Govindachari T R., Suresh G, Gopalakrishnan G. Antifungal activity of some tetranortriterpenoids. Fitoterapia, 2000, 71: 317-320
41. Hasegawa S. Limonoids in Citrus seeds: Origin and relative concentration. J. Agric. Food Chem., 1980, 28:922-925
42. Hasegawa S, Bennett R D, . Limonoid glucosides in Citrus. Phytochemistry, 1989, 28(6): 1717-1720
43. Herman Z, Fong C H, Ou P, Hasegawa S. Limonoid glucosides in orange juices by HPLC. J. Agric. Food Chem., 1990, 38:1860-1861
44. Hasegawa S, Ou P, Fong C H, Herman Z, Charles W, Coggins Jr, Darwin R A. Change in the limonoate A-ring lactone and limonin17-β-D-glucopyranoside content of navel oranges during fruit growth and matruation. J. Agric. Food Chem., 1991, 39:262-265
45. Hasegawa S, Berhow M A. Analysis of limonoids by thin-layer chromatography. In: Berhow M A. Hasegawa S. Manners G D eds., Citrus limonoids: Functional chemicals in agriculture and foods. American chemical society, Washington, DC., 2000. 31-59
46. Haswgawa S. Biochemistry of limonoids in Citrus. In: Berhow M A. Hasegawa S. Manners G D eds., Citrus Limonoids: Functional Chemicals in Agriculture and Foods. American Chemical Society, Washington, DC., 2000. 21
47. Jack D. Citrus compound-ready to help your body. Agricultural research, 2005, 2: 16-17
48. Jitpukdeebodintra S, Chantachum S, Ratanaphan A. Stability oflimonin from lime seeds. Electron. Environ. Agric. Food Chem., 2005, 4(3): 217-223
49. Kelly C, Jewell C, O'Brien N M. The effect of dietary supplementation with the citrus limonoids, limonin and nomilin on xenobiotic-metabolizing enzymes in the liver and small intestine of the rat. Nutrition Research, 2003, 23:681-690
50. Lam L K T, Li Y, Hasegawa S. Effect of citrus limonoids on glutathione s-transferase activity in mice. J. Agric. Food Chem., 1989,37:878-880
51. Liminoids strongly inhibit HIV protease activity[N]. Health & Medicine Week, 2004, 2/9
52. Maier V P, Brewster L C, Hsu A C. Specific thin-layer chromatography assay of limonin. J. Agric. Food Chem. , 1973, 18:250-252
53. Maier V P, Bennett R D, Hasegawa S. Limonin and other limonoids. In: Maier V P ed., Citrus Science and technology. INC., A Ⅵ Publishing company, 1977, 1: 355-396
54. Miller E G, Gonzales-Sanders A P, Couvillon A M. Inhibition of hamster buccal pouch carcinogenesis by limonin .17-β-D-glucopyranoside. Nutrition Cancer. 1992, 17(1): 1-7
55. Mendel M J, Alford A R, Rajab M S. Relationship of citrus limonoid structure to feeding deterrence against fall armyworm (Lepidoptera: Noetuidae) larvae. Environmental Entomology, 1993, 22(1): 167-173
56. Moodley V E, Mulholland D A, Raynor M W. Mieellar eleetrokinetie capillary chromatography of limonoid glucosides from citrus seeds. J. Chromatogr. A, 1995, 718:187-193
57. MeIntosh C A, Mansell R L. Three-dimensional distribution of limonin, limonoate A-ring monolaetone, and narigin in the fruit tissues of three varieties of Citrus paradise. J. Agric. Food Chem., 1997, 45:2876-2883
58. Matsuda H, Yoshikawa M, Iinuma M, Kubo M. Antinociceptive and antiinflammatory activites of limonin isolated from the fruits of Evodia rutaecarpa Var. bodinied. Planta Med., 1998, 64(4): 339-342
59. Manners G D. Hasegawa S. A new normal phase liquid chromatographic method for the analysis of limonoids in Citrus. Phytochem. Anal., 1999, 10:76-81
60. Murray K D, Hasegawa S, Alford A R. Antifeedant activity of citrus limonoids against Colorado potato beetle: Comparison of aglycones and glucosides. Entomologia Experimentalis Applicata, 1999, 92(3): 331-334
61. Miyake M, Shimoda M, Osajima Y, Inaba N, Ayano S, Ozaki Y, Hasegawa S. Extraction and recovery of limonoids with the supercdtical carbon dioxide micro-bubble method. A CS Symp Ser., 2000, 758: 96-106
62. Moriguchi T, Kita M, Hasegawa S, Omura M. Molecular approach to citrus flavonoid and limonoid biosynthesis. J. Food, Agriculture & Environment. 2003, 1(1): 22-25
63. Manners G D, Breksa Ⅲ A P. Identifying citrus limonoid aglycones by HPLC-EI/MS and HPLC-APCI/MS. Phytochemical Analysis, 2004, 15(6): 372-381
64. Nawwar M A M, Marzouk M S, Nigge W. High-performance liquid chromatographic/Electrospray ionization mass spectrometric screening for polyhenolic compounds of Epilobium hirostum-the structure of the unique ellagitannin epilobamide-A. Mass Spectrometry, 1997, 32: 645-654
65. Ozaki Y, Fong C H, Herman Z, Maeda H, Miyake M, Ieuku Y, Hasegawa S. Limonoid glucosides in Citrus seeds. Biol. Chem., 1991, 55(1): 137-141
66. Ohta H, Chi H Fong, Berhow M, Hasegawa S. Thin-layer and high-performance liquid chromatographic analyses of limonoids and limonoid glucosides in Citrus seeds. J. Chromatogr., 1993, 639:295-302
67. Ozaki Y, Ayano S, Inaba N, Miyake M, Bernow Mark A, Hasegawa S. Limonoid glucosides in fruit, juice and processing by-products of Satsuma Mandarin (Citrus unshiu Marcov.). Food Sci., 1995, 60(1): 186-189
68. Poulose SM, Harris E D. Citrus limonoids induce apoptosis in human neuroblastoma cells and have radical scavenging activity. Nutrition, 2005, 135(4): 870-877
69. Rouseff R L, Fisher J F. Determination of limonin and related limonoids in citrus juices by high performance liquid chromatography. Anal. Chem., 1980, 521: 228-1233
70. Rorseff R L, NaGY S. Distribution of limonoids in Citrus seeds. Phytochemistry, 1982, 21:85-90
71. Raman G, Cho M, Brodbelt J S. Isolation and purification of closely related Citrus limonoid glucosides by flash chromatography. Phytochemical Analysis, 2005, 16(3): 155-160
72. Shaw P E, Wilson C W. A rapid method for the determination of limonin in citrus juices by high performance liquid chromatography. J. Food Sci. , 1984, 49: 1216-1218
73. Shaw P E. The use of a simple solvent optimization program to improve separation of limonin in citrus juices. J. Chromatogr. Sci., 1986, 24:364-366
74. Sawabe A, Morita M, Kiso T, Kishine H, Ohtsubo Y, Minematsu T, Matsubara Y, Okamoto T. Isolation and characterization of new limonoid glycosides from Citrus unshiu peels. Carbohydrate Research, 1999, 315:142-147
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