南瓜黄色素中叶黄素的纯化及特性研究
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摘要
南瓜(Cucurbiat moschata Duch)，为葫芦科南瓜属植物的果实，栽培简单，产量高，易于贮运，而且营养丰富，深受人们的喜爱。本研究以我国主要栽培种的黄狼南瓜品种为原料，从其粉体中提出天然食用黄色素，对其组分分析，结果发现南瓜黄色素中主要含有三种类胡萝卜素：叶黄素、β-胡萝卜素和α-胡萝卜素。叶黄素对老年性黄斑变性病(AND)具有良好的预防作用，通过过滤蓝光和清除自由基而对眼睛具有良好的保护作用，有研究还显示，叶黄素对白内障和癌症都有预防作用。本研究目的在于寻找南瓜黄色素的最佳提取工艺，而后从所制备的南瓜黄色素粗品中分离纯化出叶黄素，并初步探讨其清除羟自由基和抑制猪油自氧化的能力，为南瓜综合利用提供可靠的理论依据。本研究的主要结果如下：
首先对苯、氯仿、二氯甲烷、环己烷、石油醚、乙醚、丙酮、四氢呋哺、乙醇九种溶剂的提取效果进行对比实验，其效果依次为：四氢呋喃＞氯仿＞二氯甲烷＞乙醇＞乙醚＞苯＞石油醚＞环己烷＞丙酮，而后对混合溶剂优选，结果以四氢呋喃-乙醇的提取效果最佳，最后经过正交实验，结果显示以四氢呋喃-乙醇(3：1)为提取溶剂，温度为20℃，料液比为1：5，超声波辅助提取40min连续提取五次为最佳提取工艺，得到的南瓜黄色素的提取率为4.94％。
对南瓜色素皂化品进行组分分析，HPLC条件为Waters 510泵，20μl微量进样器，Waters996检测器，Millenium色谱数据分析系统，大连依利特科学仪器有限公司提供的C_(18)色谱柱(150mm×4.6mm，5 μm)，流动相为甲醇-二氯甲烷-乙腈-水(38：32：29：1，V／V)，检测波长450nm，流速0.6ml／min，结果发现其中主要含有三种成分：叶黄素、β-胡萝卜素和α-胡萝卜素，其中叶黄素含量为27.364mg／g，β-胡萝卜素的含量为6.802mg／g。利用叶黄素与胡萝卜素存在结构上的差异以及在正己烷中的溶解度的差异，对南瓜色素中的叶黄素进行分离纯化，由HPLC分析结果显示，叶黄素纯化后得率为80.5～86.3％，纯度为90.5～94.6％。
对南瓜色素的理化特性进行研究表明，该色素对大多数金属离子是稳定的，但三价的铁离子会使该色素遭到破坏，当有Ba~(2+)存在时会产生不明沉淀，此外，
该色素对pH和防腐剂都比较稳定，温度在不超过100’C时对色素的破坏作用也
不大，但高温处理则使其组分分解，南瓜色素对光照十分敏感。分光光度法测得
皂化品中总类胡萝卜素含量为29.22%，粗品中含量为5.07%。
采用抗坏血酸一CuZ十一H八一酵母体系产生的经自由基为体外模型，对制备产品
清除轻自由基的能力进行研究，结果发现南瓜黄色素的皂化品和纯化碑能有效
清除轻自由基，最佳的浓度为0.Zmg/m1。在猪油自氧化实验中，在低浓度范围
(<0.2%)南瓜色素同样表现出了良好的抑制作用，而且在不超过VC浓度的时候
与VC具有良好的协同抗氧化作用。·
综上所述，南瓜黄色素为天然类胡萝卜素，从南瓜中提取南瓜黄色素为其综
合利用提供了一条有效的途径，尤其从中制备分离出叶黄素开发高档产品提供了
良好的理论依据。
Pumpkin (Cucurbiat moschata Duch) is the fruit of cucurbitaceous plants. Due to its easy growth, high yield, long shelf life and abundant nutriments, pumpkin is very popular in China. In this study, Chinese Huanglang pumpkin was selected as raw material. After the composition analysis of the natural food yellow pigment extracted from the pumpkin powder, the result indicated that there were three major carotenoids: a -carotene, B -carotene and lutein. Lutein can effectively reduce the risks of age-related macular degeneration (AMD). By filtering blue light and quenching free radicals, lutein protects against cataracts and cancers. The extraction processing of pumpkin yellow pigment from pumpkin powder was optimized. Besides, a simple method for isolation and purification of lutein from pumpkin yellow pigment was developed. The stabilities of the pigment were conducted. To testify its function, the scavenging free radical activities were evaluated. All the studies provide the basic theory for the general utili
zation of pumpkin. The results were shown as follows:
First of all, the extractability of 9 organic solvents were compared by the wavelengh scanning. The sequence from the better to the worse was: tetrahydrofuran (THF) > chloroform > dichloroform > ethanol > ethyl ether > benzene > petroleum ether > cyclohexane > acetone. Furthermore, when ethanol was added to THF, the OD450nm value of the extract increased rapidly. Taking the factors of temperature, organic agent and ratio of material and solvent for combined experiment, the best extraction processing was as follows: THF-EtOH (3:1,v/v) as solvent, temperature at 20℃, ratio at 1:5. Ultrasonic processing for 40min, repeating for 5 times, can improve the extraction processing. Evaporating solvent of the extract at low temperature (<40℃), the last yield of pumpkin yellow pigment was 4.94%.
After saponification of the pigment, the extract was analysed by HPLC.
The HPLC system used throughout this study consist of a Waters 510 pump (Waters, Milford, MA), a sample injector with a 20ul loop, and a Waters 996 photodiode array detector, Millenium chromatography data system. The column used was a reversed phase ultrasphere C18 column. The mobile phase was methanol/dicholomethane/acetonitrile/water (38:32:29:1,v/v), and the flow rate was 0.6ml/min. The fluent was monitored at 450nm. The chromatography showed that there were three carotenoids: a -carotene, b -carotene and lutein. The contents of lutein and B -carotene in the pigment were 27.364mg/g and 6.802mg/g respectively. Due to the solubility of lutein in hexane, lutein was isolated and purified from the saponified extract. By HPLC analysis, the average yield of lutein was 85.4% and the purity was 92.5%.
The pigment was stable to pH, preservatives, temperature not more than 100癈 and most metal ions. When there was Fe3+, it was destroyed badly. Otherwise, Ba2+ can produce the undefined precipitate. The pigment was sensitive to sunlight and high temperature (>100癈). Spectrophotometer measuring, the total content of caroteinoids in saponified product was 29.22%, and in the crude extract was 5.07%.
Using VC-CUSO4-H2O2 system to study its scavenging activity, the result indicated that both the saponified extract and the purified lutein could quench the hydroxyl radical ( OH). The best concentration of scavenging was 0.2mg/ml. In the lipid self-oxidation experiment, the product showed a strong inhibition of oxidant at low concentration (<0.2%). Furthermore, VC can enhance the antioxidant ability of the pigment.
In general, pumpkin yellow pigment was natural carotenoids. Extracting the pigment from pumpkin powder, especially isolation and purification of lutein, provide a new way for developing pumpkin products.
首先对苯、氯仿、二氯甲烷、环己烷、石油醚、乙醚、丙酮、四氢呋哺、乙醇九种溶剂的提取效果进行对比实验，其效果依次为：四氢呋喃＞氯仿＞二氯甲烷＞乙醇＞乙醚＞苯＞石油醚＞环己烷＞丙酮，而后对混合溶剂优选，结果以四氢呋喃-乙醇的提取效果最佳，最后经过正交实验，结果显示以四氢呋喃-乙醇(3：1)为提取溶剂，温度为20℃，料液比为1：5，超声波辅助提取40min连续提取五次为最佳提取工艺，得到的南瓜黄色素的提取率为4.94％。
对南瓜色素皂化品进行组分分析，HPLC条件为Waters 510泵，20μl微量进样器，Waters996检测器，Millenium色谱数据分析系统，大连依利特科学仪器有限公司提供的C_(18)色谱柱(150mm×4.6mm，5 μm)，流动相为甲醇-二氯甲烷-乙腈-水(38：32：29：1，V／V)，检测波长450nm，流速0.6ml／min，结果发现其中主要含有三种成分：叶黄素、β-胡萝卜素和α-胡萝卜素，其中叶黄素含量为27.364mg／g，β-胡萝卜素的含量为6.802mg／g。利用叶黄素与胡萝卜素存在结构上的差异以及在正己烷中的溶解度的差异，对南瓜色素中的叶黄素进行分离纯化，由HPLC分析结果显示，叶黄素纯化后得率为80.5～86.3％，纯度为90.5～94.6％。
对南瓜色素的理化特性进行研究表明，该色素对大多数金属离子是稳定的，但三价的铁离子会使该色素遭到破坏，当有Ba~(2+)存在时会产生不明沉淀，此外，
该色素对pH和防腐剂都比较稳定，温度在不超过100’C时对色素的破坏作用也
不大，但高温处理则使其组分分解，南瓜色素对光照十分敏感。分光光度法测得
皂化品中总类胡萝卜素含量为29.22%，粗品中含量为5.07%。
采用抗坏血酸一CuZ十一H八一酵母体系产生的经自由基为体外模型，对制备产品
清除轻自由基的能力进行研究，结果发现南瓜黄色素的皂化品和纯化碑能有效
清除轻自由基，最佳的浓度为0.Zmg/m1。在猪油自氧化实验中，在低浓度范围
(<0.2%)南瓜色素同样表现出了良好的抑制作用，而且在不超过VC浓度的时候
与VC具有良好的协同抗氧化作用。·
综上所述，南瓜黄色素为天然类胡萝卜素，从南瓜中提取南瓜黄色素为其综
合利用提供了一条有效的途径，尤其从中制备分离出叶黄素开发高档产品提供了
良好的理论依据。
Pumpkin (Cucurbiat moschata Duch) is the fruit of cucurbitaceous plants. Due to its easy growth, high yield, long shelf life and abundant nutriments, pumpkin is very popular in China. In this study, Chinese Huanglang pumpkin was selected as raw material. After the composition analysis of the natural food yellow pigment extracted from the pumpkin powder, the result indicated that there were three major carotenoids: a -carotene, B -carotene and lutein. Lutein can effectively reduce the risks of age-related macular degeneration (AMD). By filtering blue light and quenching free radicals, lutein protects against cataracts and cancers. The extraction processing of pumpkin yellow pigment from pumpkin powder was optimized. Besides, a simple method for isolation and purification of lutein from pumpkin yellow pigment was developed. The stabilities of the pigment were conducted. To testify its function, the scavenging free radical activities were evaluated. All the studies provide the basic theory for the general utili
zation of pumpkin. The results were shown as follows:
First of all, the extractability of 9 organic solvents were compared by the wavelengh scanning. The sequence from the better to the worse was: tetrahydrofuran (THF) > chloroform > dichloroform > ethanol > ethyl ether > benzene > petroleum ether > cyclohexane > acetone. Furthermore, when ethanol was added to THF, the OD450nm value of the extract increased rapidly. Taking the factors of temperature, organic agent and ratio of material and solvent for combined experiment, the best extraction processing was as follows: THF-EtOH (3:1,v/v) as solvent, temperature at 20℃, ratio at 1:5. Ultrasonic processing for 40min, repeating for 5 times, can improve the extraction processing. Evaporating solvent of the extract at low temperature (<40℃), the last yield of pumpkin yellow pigment was 4.94%.
After saponification of the pigment, the extract was analysed by HPLC.
The HPLC system used throughout this study consist of a Waters 510 pump (Waters, Milford, MA), a sample injector with a 20ul loop, and a Waters 996 photodiode array detector, Millenium chromatography data system. The column used was a reversed phase ultrasphere C18 column. The mobile phase was methanol/dicholomethane/acetonitrile/water (38:32:29:1,v/v), and the flow rate was 0.6ml/min. The fluent was monitored at 450nm. The chromatography showed that there were three carotenoids: a -carotene, b -carotene and lutein. The contents of lutein and B -carotene in the pigment were 27.364mg/g and 6.802mg/g respectively. Due to the solubility of lutein in hexane, lutein was isolated and purified from the saponified extract. By HPLC analysis, the average yield of lutein was 85.4% and the purity was 92.5%.
The pigment was stable to pH, preservatives, temperature not more than 100癈 and most metal ions. When there was Fe3+, it was destroyed badly. Otherwise, Ba2+ can produce the undefined precipitate. The pigment was sensitive to sunlight and high temperature (>100癈). Spectrophotometer measuring, the total content of caroteinoids in saponified product was 29.22%, and in the crude extract was 5.07%.
Using VC-CUSO4-H2O2 system to study its scavenging activity, the result indicated that both the saponified extract and the purified lutein could quench the hydroxyl radical ( OH). The best concentration of scavenging was 0.2mg/ml. In the lipid self-oxidation experiment, the product showed a strong inhibition of oxidant at low concentration (<0.2%). Furthermore, VC can enhance the antioxidant ability of the pigment.
In general, pumpkin yellow pigment was natural carotenoids. Extracting the pigment from pumpkin powder, especially isolation and purification of lutein, provide a new way for developing pumpkin products.
引文
[1]白天珠 张福成功率超声在食品、医药等领域的应用声学与电于工程 1990，18(2)：37～40
[2]陈仁惇 编著《营养保健食品》北京：中国轻工业出版社 2001．1 133—134
[3]陈波 周光宏 曹光辛 类胡萝卜素在动物体内的代谢 国外畜牧科技 1998，25(5)：2～5
[3]陈智民，南瓜粉降血糖、将血压作用的人体研究[J]．江西中医药，1996：825～827
[4]陈学平 主编 《果蔬产品加工工艺学》中国农业出版社 1997．5：13页
[5]杜桂彩，滕大为，李荣贵，郭群群 金盏菊中叶黄素的分离纯化及高效液相色谱的测定方法精细化工 2001 18(8)：467～469
[6]董文宾，南瓜精口服液的工艺条件研究[J]．食品工业，1997，(1)：47～48
[7]樊开明，郑远旗，李建吾 食晶添加剂 四川科学技术出版社 1987．6 86～92
[8]范镇基，南瓜子氨酸的提取及其用途[J]．广东科技，1998(6)：24
[9]冯孝义．张小荣．冯向荣，微量元素铬与健康，生物学通报，1995，30(120)：13～14
[10]高蓝 李浩明 类胡萝卜素与癌症的化学预防 中草药 1998 29(5)：346—348
[11]苟劲 叶黄素及其应用研究重庆师范学院学报(自然科学版) 2002 19(2)：56-59
[12]果秀敏，翟彤宇 牛延锋 薄层色谱法分离菠菜叶片中色素的研究河北农业大学学报2000，23(2)：101～104
[13]甘长海超声波清洗过滤器技术过滤与分离2001，11(2)：20—21
[14]韩雅珊 类胡萝卜素的功能研究进展中国农业大学学报 1999．4(1)：5—9
[15]贺小琼．陈彦红．肖建春等，南瓜粉开发及营养成份分析．昆明医学院学报，1999，20(3)：46～48
[16]胡天喜，陈杞，陈克明等 发光分析与医学 华东师范大学出版社 1990，2：48～60
[17]黄文 谢笔钧 付勇 刘红东 彭光华 白果中类胡萝卜素的提取江苏食品与发酵2001，107(4)：3～5
[18]金桂英．贾芬，南瓜粉的营养、药用价值与开发利用．福建农业科技，1998 (2)：26～27
[19]孔庆胜．王彦英．蔣滢，南瓜多糖的分离、纯化及降血糖脂作用[J]．中国生化药物杂志．2000，21(3)；130～132
[20]蔺定运 食用色素的识别与应用 中国食品出版社 1987．6 53～55
[21]酆惠芬 超声去脂原理和高效换能器的设计 应用声学 2001，20(6)：41～44
[22]李丙东，刘宜生，王长林，南瓜属蔬菜生物学基础研究概况及育种进展，中国蔬菜，1996(6)：48～50
[23]刘钟栋 编著《食品添加剂原理及应用技术》(第二版)中国轻工业出版社 2000．4
[24]刘晓艳 丘奉球 刘石生 胡爱军 超声对细胞膜通透性的影响及应用应用声学 2002，21(2)：26～29
[25]刘璇 类胡萝卜素的药用价值国外医学中医中药分册 1995，17(3)：18～19
[26]刘钟栋 编著《食品添加剂原理及应用技术》(第二版)中国轻工业出版社 2000．4
[27]姜文武 单志萍 孟好 微生物生产的类胡萝卜素及其工业化食品与发酵工业 1999，25(3)46～50
[28]马钟锦，编译 含叶黄素的食物对视力有益美食营养(中国食品)2000．9
[29]马自超，庞业珍编著《天然食用色素化学及生产工艺》中国轻工业出版社 1994．9
[30]潘向荣，硕士论文《杨梅抗氧化特性研究》2002．5：23～25
[31]日本厚生省环境卫生局食品化学课程编《食品中添加剂的分析方法》 中国标准出版社1988．2：504—512
[32]《世界精细化工手册》化学工业部科学技术情报研究所 太原 1986．5：264～265
[33]宋曙辉，薛颖 高效液相色谱法测定蔬菜中的类胡萝卜素华北农学报 2001，16(3)；92—97
[34]石永峰 国外食品天然色素的研究进展粮油与食品工业 1996，(4)：23～27
[35]王业勤，李勤生，编著天然类胡萝卜素——研究进展、生产、应用 北京：中国医药科技出版社 1997，3：220～233
[36]王璋 许时婴 汤坚《食品化学》中国轻二正业出版社 2001．1
[37]王强．韩雅珊．广田才之，食品多种胡萝卜素的测定(HPLC 法)[J]．营养学报，1997，19(2)：212～215
[38]王威 王春利 常用食用防腐剂对食用天然色素影响的研究食品与发酵工业2001．21(2)：38～41
[39]王琳，南瓜治疗糖尿病临床观察报告．东北师大学报自然科学版，1993(4)：83～84
[40]吴寿金．李德玉．降血糖植物多糖的研究概况．中草药，1992，23(40)：549～554
[41]武维振．王兰．刘红丽，天然食用色素南瓜黄的制备和性质研究[J]．郑州粮食学院学报，1996，17(2)：25～29
[42]武维振．王兰．刘红丽，天然食用色素南瓜黄的稳定性及应用研究[J]．郑州粮食学院报，1996，17(3)：28～32
[43]熊学敏．石扬．康明等，南瓜多糖有效部位的提取分离及降糖作用的研究[J]．中草药，2000，22(8)：563～565
[44]叶青，裘兆蓉，王车礼，郭本炎，周菊欢 柑桔皮中桔黄色素的提取广东化工 1999(7)：40～41
[45]袁易全．近代超声原理与应用．南京南京大学出版社 1996．108—168．
[46]赵传集，南瓜产地小考，农业考古 1987(2)：299～300
[47]赵哲勋 天然色素稳定剂研究农牧产品开发 1999．(4)：17～18
[48]赵文恩，韩雅珊．类胡萝卜素清除活性氧自由基的机理，化学通报，1999(4)：25～27
[49]张建民番茄红素——西红柿中提取的类胡萝卜素中国制药信息 2000．16(8)：22～27
[50]张建华，胡飞，南瓜色素的提取及稳定性研究 上海农学院学报 2000．18(2)：142～144
[51]张水成 南瓜果肉色素的提取及稳定性的研究 河南师范大学学报(自然科学版)2000，28(3)：133～134
[52]张莲彩 李娜 天然色素的发展及其在食品中的应用天津轻工 1990．48(4)：13～19
[53]张芳．蔣作明．章恩明，南瓜的功能特性及其在食品工业总的应用[J]．食品工业科技，2000，21(6)：62～64
[54]张之佳．洪伯铿．张拥军，超细南瓜粉对家免试验性糖尿病作用的研究[J]．中国粮油学报，1998，13(2)：52～56
[55]张拥军．姚惠源，两种不同品种的南瓜多糖降糖效果研究[J]．食品科学 2002，23(2)：118～120
[56]张建华．胡飞．南瓜色素的提取及稳定性研究，上海农学院学报，2000．18(2)：42～43
[57]郑鹏然．周树南主编，食品卫生全书[M]．北京：红旗出版社，1996：825～827
[58]中国预防医学院科学院 营养与食品卫生研究所编著《食物成分表》(全国代表值)人民卫生出版社 1993．3
[59]中国食品添加剂生产应用工业协会主编《食品添加剂分析检测手册》中国轻工业出版社 1995．5
[60]左兴一．马少怀．南瓜酸牛乳(适于糖尿病人)的制造．食品工业科技，2002，23(4)：9～10
[61]A.Hager, in Patierchromatographie in der Botanik, ed. by H.F.Lindskens (Springer, Heidelberg, 1959):218
[62]"Antioxidant status and neovascular age-related macular degeneration."Eye disease Case-Control Study Group(1993). Arch Ophthalmol 111(1):104-109
[63]A.Subagio,N.Morta,no effect of esterification with fatty acid on antioxidant activity of lutein, Food Research International 34(2001):315-320
[64]B.H. Davies, in Chemistry and Biochemistry of Plant Pigments, ed. by T.W. Goodwin (Academic Press, London 1965): 489
[65]Bone, R.A.,J.T.Landrum, et al.(1993)."Stereochemistry of the human macular carotenoids." Invest Ophthalmol Vis Sci 34(6):2033-40
[66]Bone,R.A.,J.T.Landrum, et al.(2001)."macular pigment in donor eyes with and without AMD: A case-control study" Invest Ophthalmol Vis Sci 42(1):235-240
[67]Bone,R.A.,J.T.Landrum, et al. (2000)."Lutein and zeaxanthin in the eyes, serum and diet of human subjects."Exp Eye Res 71(3):239-245
[68]Beatty, S., M.Boulton, et at.(1999). "Macular pigment and age related macular degeneration." Br J Ophthalmol 83(7):867-877
[69]Christen,W.G.,R.J.Glynn, et al. (1996). "A prospective study of cigarette smoking and risk of age-related macular degeneration in men." Jama 276(14):1147-1151
[70]DIETMAR E. BRETTHAUPT, URSULA WlRT, AMENEH BAMEDI Differentiation between lutein Monoester Regioisomers and Detection of Lutein Diesters from Marigold Flowers (Tagetes erecta L.) and Several Fruits by Liquid Chromatography-Mass
[71]Spectrometry J. Agric. Food Chem. 2002,50,66-70
[72]Drummond, J.C. The nomenclature of the so called accessory food factors(vitamins). Biochem. J. 14:660-674;1920
[73]Federocl Khachik, Andrea Steck, Hanspeter Pfander. Isolation and structural Elucidation of (13Z,13'Z,3R,3'R,6'R)-Lutein from Marigold flowers, Kale and Human Plasma J. Agric. Food Chem. 1999,47,455-461
[74]Federocl Khachik, Andrea Steck, Hanspeter Pfander. Isolation and structural Elucidation of (13Z,13'Z,3R,3'R,6'R)-Lutein from Marigold flowers, Kale and Human Plasma J. Agric. Food Chem. 1999,47,455-461
[75]Foote, C.W. ; Denny, R.W. Chemistry of singlet oxygen. Ⅶ. Quenching by β -carotene. J.Am. Chem. Soc. 90:6233-6235; 1968
[76]Garry J.Handelman .The evolving Role of Carotenoids in Human biochemistry. Nutrition in Elsevier Science Inc.2001 17:818-822
[77]Gerely A. Csiktusnadi Kiss,Esther Forgacs etc.(2000) Optimisation of the microwave-assisted extraction of pigments from paprika(Capsicum annuumm L.)powders. Journal of Chromatography A,(2000):41-49
[77]Hammond, B.R.,B.R.Wooten, et al.(1998). "Preservation of visual sensitivity of older subjects: association with macular pigment density."Invest Ophthalmol Vis Sci 39(12):397-406
[78]Hammond, B.R., B.R.Wooten, et al. (1996). "Cigarette smoking and retinal carotenoids: implications for age-related macular degeneration." Vision Res 36(18):3003-3009
[79]Hammond, B.R., K.Fuld, et al.(1995). "Macular pigment density in monozygotic twins." Invest Ophthalmol Vis Sci 36(12):2531-2541
[80]HUA-BIN LI, YUE JIANG, FENG CHEN Isolation and Purification of Lutein from the
Microalga Chlorella vuagaris by Extraction after Saponification J. Agic. Food Chem. 2002,50,1071-1072
[81]J. Agric.Food Chem. 1986,34,603～616
[82]Jordi Oliver, Andreu Palou ,Chromatographic determination of carotenoids in foods Journal of Chromatography A 881(2000)542-555
[83]Khachik,F.,G. R.Beecher, et al.(1995). "Lutein, lycopene, and their oxidantive metabolites in chemoprevention of cancer." J Cell Biochem Suppl 22:236-246
[84]Krinsky, N. I,; X. D. ; Tang, G,; Russell, R. M. Mechanism of carotenoid·cleavage to retinoids: Ann. N.Y. Acad .Sci. 691:167-176;1993
[85]Landrum,J.T.,Y.Chen, et al.(2000). "Serum and macular pigment response to 2.4mgDosage of lutein." ARVO41(4):S60
[86]L.Zechmeister, quoted by W.Katter, Konstitution und Vorkommen der organischen Pflanzenstoffe, Birkhauser, Basle, 1958:716
[87]M.Murkovic, U.Mlleder and H.Neunteufl, Carotenoid Content in Different Varieties of Pumpkins, JOURNAL OF FOOD COMPOSITION AND ANALYSIS (2002)15,633-638
[88]Moore, T. Vitamin A. Amsterdam : Elsevire publishing company ,1957
[89]Neuringer, M., E.J.Johnson, et al.(2001)."Supplementation of carotenoid-depleted rhesus monkeys with lutein or zeaxinthin: Effects on serum and adipose tissue carotenoids and macular pigment." FASEB 24(4):S244
[90]OTTA ISLER, HUGO GUTMANN and ULRICH SOLMS, Carotenoids (BIRKHAUSER VERLAG BASEL UND STUTTGART, 1971): 64
[91]Patt,S.(1999). "Dietary prevention of age-related macular degeneration." J Am Optom Assoc 70(1):39-47
[92]Piere Astorg.(1997)Food crotenoids and cancer prevention:An overview of current research In Trends in Food Scinence&Technology December 1997,8:406-412
[93]Rapp, L.M.,S.S.Maple, et al.(2000). "Lutein and zeaxanthin concentrations in rod outer segment memgbranes from perifoveal and peripheral human retina." Invest Ophthalmol Vis Sci 41 (5):1200-1209
[94]Piere Astorg.(1997)Food crotenoids and cancer prevention:An overview of current research In Trends in Food Scinence&Technology December 1997,8:406-412
[95]Richer, S.(1999). "A protocol for the evaluation and treatment of atrophic age-related macular degeneration." J Am Optom Assoc 70(1):13-23
[96]Richer, S.(1996). "Atrophic armd—a nutrition responsive chronic disease" J Am Optom Assoc67(1):6-10
[97]Sandra Naranjo-Modad, Agustin López-Mungui a, et al, Solubility of Purified Lutein Diesters Obtained from Rageies erecta in Supercritical CO_2 and the Effect of solvent Modifiers J. Agric. Food Chem. 2000,48,5640-5642
[98]Seddon, J.M., W.C.Willett, et al.(1996). "A prospective-study of cigarette smoking and age-related macular degeneration in women." Jama 276(14): 1147-1146
[99]Snodderly, D.M.(1995). "Evidence for protection against age-related macular degeneration by carotenoids and antioxidant vitamins." Am J Clin Nut 62(6):1448S-1461S
[100]Seddon, J.M., U.A.Ajani, et al. (1994). "Dietary carotenoids, vitamins A, C and E, and advanced age-related macular degeneration." Eye Disease Case-Control Study Group Jama 272(18):1413-1420
[101]Sundelin, S.P. and S.E.Nisson(2001). "Lipofuscin-formation in retinal pigment epithelial cells is reduced by antioxidants." Free Radic Biol Med 31(2):217-225
[102]Toshiro Watanabe, Shigeru Terabe, Analysis of natural food pigments by capillary electrophoresis Journal of Chromatography A, 880(2000)311-322
[103]T.W. Goodwin, in Chemistry and Biochemistry of Plant Pigments, ed. by T.W.Goodwin, (Academic Press, London 1965):127
[104]VINCE P. PALACE, NEELAM KHAPER, QINING QIN, PAWAN K. SINGAL ANTIOXlDANT POTENTIALS OF VITAMIN A AND CAROTENOIDS AND THIR RELEAVANCE TO HEART DISEASE Free Radical Biology & Medicine 1999 vol.26:746-761
[105]Y.C.Tang, B.H.Chen, Pigment change of freeze-dried carotenoid powder during storage, Food Chemistry 69(2000): 11-17
[106]Yemelyanov, A.Y., N.B.Katz, et al.(2001). "Ligand-binding characterization of xanthophylls carotenoids to soluilized membrane proteins drived from human retina," Exp Eye Res 72(4):381-392
[2]陈仁惇 编著《营养保健食品》北京：中国轻工业出版社 2001．1 133—134
[3]陈波 周光宏 曹光辛 类胡萝卜素在动物体内的代谢 国外畜牧科技 1998，25(5)：2～5
[3]陈智民，南瓜粉降血糖、将血压作用的人体研究[J]．江西中医药，1996：825～827
[4]陈学平 主编 《果蔬产品加工工艺学》中国农业出版社 1997．5：13页
[5]杜桂彩，滕大为，李荣贵，郭群群 金盏菊中叶黄素的分离纯化及高效液相色谱的测定方法精细化工 2001 18(8)：467～469
[6]董文宾，南瓜精口服液的工艺条件研究[J]．食品工业，1997，(1)：47～48
[7]樊开明，郑远旗，李建吾 食晶添加剂 四川科学技术出版社 1987．6 86～92
[8]范镇基，南瓜子氨酸的提取及其用途[J]．广东科技，1998(6)：24
[9]冯孝义．张小荣．冯向荣，微量元素铬与健康，生物学通报，1995，30(120)：13～14
[10]高蓝 李浩明 类胡萝卜素与癌症的化学预防 中草药 1998 29(5)：346—348
[11]苟劲 叶黄素及其应用研究重庆师范学院学报(自然科学版) 2002 19(2)：56-59
[12]果秀敏，翟彤宇 牛延锋 薄层色谱法分离菠菜叶片中色素的研究河北农业大学学报2000，23(2)：101～104
[13]甘长海超声波清洗过滤器技术过滤与分离2001，11(2)：20—21
[14]韩雅珊 类胡萝卜素的功能研究进展中国农业大学学报 1999．4(1)：5—9
[15]贺小琼．陈彦红．肖建春等，南瓜粉开发及营养成份分析．昆明医学院学报，1999，20(3)：46～48
[16]胡天喜，陈杞，陈克明等 发光分析与医学 华东师范大学出版社 1990，2：48～60
[17]黄文 谢笔钧 付勇 刘红东 彭光华 白果中类胡萝卜素的提取江苏食品与发酵2001，107(4)：3～5
[18]金桂英．贾芬，南瓜粉的营养、药用价值与开发利用．福建农业科技，1998 (2)：26～27
[19]孔庆胜．王彦英．蔣滢，南瓜多糖的分离、纯化及降血糖脂作用[J]．中国生化药物杂志．2000，21(3)；130～132
[20]蔺定运 食用色素的识别与应用 中国食品出版社 1987．6 53～55
[21]酆惠芬 超声去脂原理和高效换能器的设计 应用声学 2001，20(6)：41～44
[22]李丙东，刘宜生，王长林，南瓜属蔬菜生物学基础研究概况及育种进展，中国蔬菜，1996(6)：48～50
[23]刘钟栋 编著《食品添加剂原理及应用技术》(第二版)中国轻工业出版社 2000．4
[24]刘晓艳 丘奉球 刘石生 胡爱军 超声对细胞膜通透性的影响及应用应用声学 2002，21(2)：26～29
[25]刘璇 类胡萝卜素的药用价值国外医学中医中药分册 1995，17(3)：18～19
[26]刘钟栋 编著《食品添加剂原理及应用技术》(第二版)中国轻工业出版社 2000．4
[27]姜文武 单志萍 孟好 微生物生产的类胡萝卜素及其工业化食品与发酵工业 1999，25(3)46～50
[28]马钟锦，编译 含叶黄素的食物对视力有益美食营养(中国食品)2000．9
[29]马自超，庞业珍编著《天然食用色素化学及生产工艺》中国轻工业出版社 1994．9
[30]潘向荣，硕士论文《杨梅抗氧化特性研究》2002．5：23～25
[31]日本厚生省环境卫生局食品化学课程编《食品中添加剂的分析方法》 中国标准出版社1988．2：504—512
[32]《世界精细化工手册》化学工业部科学技术情报研究所 太原 1986．5：264～265
[33]宋曙辉，薛颖 高效液相色谱法测定蔬菜中的类胡萝卜素华北农学报 2001，16(3)；92—97
[34]石永峰 国外食品天然色素的研究进展粮油与食品工业 1996，(4)：23～27
[35]王业勤，李勤生，编著天然类胡萝卜素——研究进展、生产、应用 北京：中国医药科技出版社 1997，3：220～233
[36]王璋 许时婴 汤坚《食品化学》中国轻二正业出版社 2001．1
[37]王强．韩雅珊．广田才之，食品多种胡萝卜素的测定(HPLC 法)[J]．营养学报，1997，19(2)：212～215
[38]王威 王春利 常用食用防腐剂对食用天然色素影响的研究食品与发酵工业2001．21(2)：38～41
[39]王琳，南瓜治疗糖尿病临床观察报告．东北师大学报自然科学版，1993(4)：83～84
[40]吴寿金．李德玉．降血糖植物多糖的研究概况．中草药，1992，23(40)：549～554
[41]武维振．王兰．刘红丽，天然食用色素南瓜黄的制备和性质研究[J]．郑州粮食学院学报，1996，17(2)：25～29
[42]武维振．王兰．刘红丽，天然食用色素南瓜黄的稳定性及应用研究[J]．郑州粮食学院报，1996，17(3)：28～32
[43]熊学敏．石扬．康明等，南瓜多糖有效部位的提取分离及降糖作用的研究[J]．中草药，2000，22(8)：563～565
[44]叶青，裘兆蓉，王车礼，郭本炎，周菊欢 柑桔皮中桔黄色素的提取广东化工 1999(7)：40～41
[45]袁易全．近代超声原理与应用．南京南京大学出版社 1996．108—168．
[46]赵传集，南瓜产地小考，农业考古 1987(2)：299～300
[47]赵哲勋 天然色素稳定剂研究农牧产品开发 1999．(4)：17～18
[48]赵文恩，韩雅珊．类胡萝卜素清除活性氧自由基的机理，化学通报，1999(4)：25～27
[49]张建民番茄红素——西红柿中提取的类胡萝卜素中国制药信息 2000．16(8)：22～27
[50]张建华，胡飞，南瓜色素的提取及稳定性研究 上海农学院学报 2000．18(2)：142～144
[51]张水成 南瓜果肉色素的提取及稳定性的研究 河南师范大学学报(自然科学版)2000，28(3)：133～134
[52]张莲彩 李娜 天然色素的发展及其在食品中的应用天津轻工 1990．48(4)：13～19
[53]张芳．蔣作明．章恩明，南瓜的功能特性及其在食品工业总的应用[J]．食品工业科技，2000，21(6)：62～64
[54]张之佳．洪伯铿．张拥军，超细南瓜粉对家免试验性糖尿病作用的研究[J]．中国粮油学报，1998，13(2)：52～56
[55]张拥军．姚惠源，两种不同品种的南瓜多糖降糖效果研究[J]．食品科学 2002，23(2)：118～120
[56]张建华．胡飞．南瓜色素的提取及稳定性研究，上海农学院学报，2000．18(2)：42～43
[57]郑鹏然．周树南主编，食品卫生全书[M]．北京：红旗出版社，1996：825～827
[58]中国预防医学院科学院 营养与食品卫生研究所编著《食物成分表》(全国代表值)人民卫生出版社 1993．3
[59]中国食品添加剂生产应用工业协会主编《食品添加剂分析检测手册》中国轻工业出版社 1995．5
[60]左兴一．马少怀．南瓜酸牛乳(适于糖尿病人)的制造．食品工业科技，2002，23(4)：9～10
[61]A.Hager, in Patierchromatographie in der Botanik, ed. by H.F.Lindskens (Springer, Heidelberg, 1959):218
[62]"Antioxidant status and neovascular age-related macular degeneration."Eye disease Case-Control Study Group(1993). Arch Ophthalmol 111(1):104-109
[63]A.Subagio,N.Morta,no effect of esterification with fatty acid on antioxidant activity of lutein, Food Research International 34(2001):315-320
[64]B.H. Davies, in Chemistry and Biochemistry of Plant Pigments, ed. by T.W. Goodwin (Academic Press, London 1965): 489
[65]Bone, R.A.,J.T.Landrum, et al.(1993)."Stereochemistry of the human macular carotenoids." Invest Ophthalmol Vis Sci 34(6):2033-40
[66]Bone,R.A.,J.T.Landrum, et al.(2001)."macular pigment in donor eyes with and without AMD: A case-control study" Invest Ophthalmol Vis Sci 42(1):235-240
[67]Bone,R.A.,J.T.Landrum, et al. (2000)."Lutein and zeaxanthin in the eyes, serum and diet of human subjects."Exp Eye Res 71(3):239-245
[68]Beatty, S., M.Boulton, et at.(1999). "Macular pigment and age related macular degeneration." Br J Ophthalmol 83(7):867-877
[69]Christen,W.G.,R.J.Glynn, et al. (1996). "A prospective study of cigarette smoking and risk of age-related macular degeneration in men." Jama 276(14):1147-1151
[70]DIETMAR E. BRETTHAUPT, URSULA WlRT, AMENEH BAMEDI Differentiation between lutein Monoester Regioisomers and Detection of Lutein Diesters from Marigold Flowers (Tagetes erecta L.) and Several Fruits by Liquid Chromatography-Mass
[71]Spectrometry J. Agric. Food Chem. 2002,50,66-70
[72]Drummond, J.C. The nomenclature of the so called accessory food factors(vitamins). Biochem. J. 14:660-674;1920
[73]Federocl Khachik, Andrea Steck, Hanspeter Pfander. Isolation and structural Elucidation of (13Z,13'Z,3R,3'R,6'R)-Lutein from Marigold flowers, Kale and Human Plasma J. Agric. Food Chem. 1999,47,455-461
[74]Federocl Khachik, Andrea Steck, Hanspeter Pfander. Isolation and structural Elucidation of (13Z,13'Z,3R,3'R,6'R)-Lutein from Marigold flowers, Kale and Human Plasma J. Agric. Food Chem. 1999,47,455-461
[75]Foote, C.W. ; Denny, R.W. Chemistry of singlet oxygen. Ⅶ. Quenching by β -carotene. J.Am. Chem. Soc. 90:6233-6235; 1968
[76]Garry J.Handelman .The evolving Role of Carotenoids in Human biochemistry. Nutrition in Elsevier Science Inc.2001 17:818-822
[77]Gerely A. Csiktusnadi Kiss,Esther Forgacs etc.(2000) Optimisation of the microwave-assisted extraction of pigments from paprika(Capsicum annuumm L.)powders. Journal of Chromatography A,(2000):41-49
[77]Hammond, B.R.,B.R.Wooten, et al.(1998). "Preservation of visual sensitivity of older subjects: association with macular pigment density."Invest Ophthalmol Vis Sci 39(12):397-406
[78]Hammond, B.R., B.R.Wooten, et al. (1996). "Cigarette smoking and retinal carotenoids: implications for age-related macular degeneration." Vision Res 36(18):3003-3009
[79]Hammond, B.R., K.Fuld, et al.(1995). "Macular pigment density in monozygotic twins." Invest Ophthalmol Vis Sci 36(12):2531-2541
[80]HUA-BIN LI, YUE JIANG, FENG CHEN Isolation and Purification of Lutein from the
Microalga Chlorella vuagaris by Extraction after Saponification J. Agic. Food Chem. 2002,50,1071-1072
[81]J. Agric.Food Chem. 1986,34,603～616
[82]Jordi Oliver, Andreu Palou ,Chromatographic determination of carotenoids in foods Journal of Chromatography A 881(2000)542-555
[83]Khachik,F.,G. R.Beecher, et al.(1995). "Lutein, lycopene, and their oxidantive metabolites in chemoprevention of cancer." J Cell Biochem Suppl 22:236-246
[84]Krinsky, N. I,; X. D. ; Tang, G,; Russell, R. M. Mechanism of carotenoid·cleavage to retinoids: Ann. N.Y. Acad .Sci. 691:167-176;1993
[85]Landrum,J.T.,Y.Chen, et al.(2000). "Serum and macular pigment response to 2.4mgDosage of lutein." ARVO41(4):S60
[86]L.Zechmeister, quoted by W.Katter, Konstitution und Vorkommen der organischen Pflanzenstoffe, Birkhauser, Basle, 1958:716
[87]M.Murkovic, U.Mlleder and H.Neunteufl, Carotenoid Content in Different Varieties of Pumpkins, JOURNAL OF FOOD COMPOSITION AND ANALYSIS (2002)15,633-638
[88]Moore, T. Vitamin A. Amsterdam : Elsevire publishing company ,1957
[89]Neuringer, M., E.J.Johnson, et al.(2001)."Supplementation of carotenoid-depleted rhesus monkeys with lutein or zeaxinthin: Effects on serum and adipose tissue carotenoids and macular pigment." FASEB 24(4):S244
[90]OTTA ISLER, HUGO GUTMANN and ULRICH SOLMS, Carotenoids (BIRKHAUSER VERLAG BASEL UND STUTTGART, 1971): 64
[91]Patt,S.(1999). "Dietary prevention of age-related macular degeneration." J Am Optom Assoc 70(1):39-47
[92]Piere Astorg.(1997)Food crotenoids and cancer prevention:An overview of current research In Trends in Food Scinence&Technology December 1997,8:406-412
[93]Rapp, L.M.,S.S.Maple, et al.(2000). "Lutein and zeaxanthin concentrations in rod outer segment memgbranes from perifoveal and peripheral human retina." Invest Ophthalmol Vis Sci 41 (5):1200-1209
[94]Piere Astorg.(1997)Food crotenoids and cancer prevention:An overview of current research In Trends in Food Scinence&Technology December 1997,8:406-412
[95]Richer, S.(1999). "A protocol for the evaluation and treatment of atrophic age-related macular degeneration." J Am Optom Assoc 70(1):13-23
[96]Richer, S.(1996). "Atrophic armd—a nutrition responsive chronic disease" J Am Optom Assoc67(1):6-10
[97]Sandra Naranjo-Modad, Agustin López-Mungui a, et al, Solubility of Purified Lutein Diesters Obtained from Rageies erecta in Supercritical CO_2 and the Effect of solvent Modifiers J. Agric. Food Chem. 2000,48,5640-5642
[98]Seddon, J.M., W.C.Willett, et al.(1996). "A prospective-study of cigarette smoking and age-related macular degeneration in women." Jama 276(14): 1147-1146
[99]Snodderly, D.M.(1995). "Evidence for protection against age-related macular degeneration by carotenoids and antioxidant vitamins." Am J Clin Nut 62(6):1448S-1461S
[100]Seddon, J.M., U.A.Ajani, et al. (1994). "Dietary carotenoids, vitamins A, C and E, and advanced age-related macular degeneration." Eye Disease Case-Control Study Group Jama 272(18):1413-1420
[101]Sundelin, S.P. and S.E.Nisson(2001). "Lipofuscin-formation in retinal pigment epithelial cells is reduced by antioxidants." Free Radic Biol Med 31(2):217-225
[102]Toshiro Watanabe, Shigeru Terabe, Analysis of natural food pigments by capillary electrophoresis Journal of Chromatography A, 880(2000)311-322
[103]T.W. Goodwin, in Chemistry and Biochemistry of Plant Pigments, ed. by T.W.Goodwin, (Academic Press, London 1965):127
[104]VINCE P. PALACE, NEELAM KHAPER, QINING QIN, PAWAN K. SINGAL ANTIOXlDANT POTENTIALS OF VITAMIN A AND CAROTENOIDS AND THIR RELEAVANCE TO HEART DISEASE Free Radical Biology & Medicine 1999 vol.26:746-761
[105]Y.C.Tang, B.H.Chen, Pigment change of freeze-dried carotenoid powder during storage, Food Chemistry 69(2000): 11-17
[106]Yemelyanov, A.Y., N.B.Katz, et al.(2001). "Ligand-binding characterization of xanthophylls carotenoids to soluilized membrane proteins drived from human retina," Exp Eye Res 72(4):381-392