天然产物的提取及其抗自由基活性的研究
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摘要
随着生活水平的日益提高，人们对饮食天然性、健康性的追求也越来越高，天然的功能性食品番茄红素、叶黄素因具有防癌、抗癌作用已经成为目前研究的热点。本论文对番茄红素、叶黄素的提取分离进行了研究，确立了有效提取番茄红素和叶黄素的工艺方法，并对获取的产品清除超氧阴离子自由基的能力进行了测定。另外，以Fenton反应为基础，对其它几种天然产物的抗羟自由基作用也进行了研究。具体工作如下：
1．番茄红素的提取工艺研究 通过番茄红素提取过程中脱水方法、溶剂提取、纯化方法等各程序中实验参数的考察，确定了有效提取番茄红素的最佳工艺条件：首先用5g食盐对20g番茄果肉进行脱水，然后加丙酮处理，所得番茄用20ml乙酸乙酯提取，提取液浓缩后得到番茄红素油树脂，通过柱层析法纯化，获得的产品吸收光谱图与文献番茄红素的标准图谱相一致，本工艺可为番茄的深度开发提供方法参考和理论依据，具有推广应用价值。
2．叶黄素的提取工艺研究 通过叶黄素提取过程中各种实验因素的考察，确定了从万寿菊中提取叶黄素的最佳工艺：6g万寿菊粉通过正己烷萃取后浓缩再用3.3％的NaOH皂化处理，可以获得0.0795g叶黄素。并对其进行了薄层层析，此研究建立的工艺体系可为通过万寿菊获取叶黄素提供方法参考，从而为万寿菊的推广种植提供理论依据。
3．通过分光光度法研究了番茄红素和叶黄素对邻苯三酚自氧化产生超氧阴离子自由基的清除作用，结果表明这两种天然色素对超氧阴离子自由基均有明显的清除效果，相同浓度的叶黄素的清除率明显高于番茄红素。
4．利用甲基紫-Fe~(2+)-H_2O_2和Fe~(2+)-邻二氮菲-H_2O_2两种反应体系研究了天然产物猕猴桃、苹果、桔子、梨、红茶、绿茶和山药对·OH的清除能力，结果表明：苹果、猕猴桃、桔子、梨的清除·OH能力依次下降，红茶和绿茶清除能力相当。两种反应体系比较，甲基紫-Fe~(2+)-H_2O_2测定灵敏度高于Fe~(2+)-邻二氮菲-H_2O_2体系。
With the increasing improvement of living standard, people become to pursuit dietary nature and health. As a kind of functional food, Lycopene and lutein are widely studied because of their usage of preventing cancer and anti-cancer. In this paper, not only extraction methods and scavenging ability on super oxide anions of lycopene and lutein were studied, but also scavenging effect on hydroxyl radical of another natural products was measured. Detailed contents are followed:
1. Extraction of lycopene from fresh tomato was investigated in order to determine its dehydration methods, extraction solvent and purification technology. 5g salts were used in the dehydration of 20g tomato pulp. 20ml Ethyl acetate was used to extract lycopene from tomato tissue after being disposed with acetone. Through above extraction technology, 0.897mg lypocene can be obtained. Lypocene-containing oleoresin were gained after condense. Saponification and colummation layer analysis were used to sublimate lycopene oleoresin. Spectrogram of lycopene products gained is agreed with its standard Spectrogram. Colummation layer analysis superior to Saponification by comparing their extraction rate.
2.We investigate the technogical conditions for extracting lutein from marigold (Tagetes erecta) and determined its optimum technology. When concentration of NaOH is 3.3%, extracting time was two hours and extraction times was two times, 0. 0795g lutein products are obtained from 6g marigolds. It was analyzed by thin-layer chromatography on silica plates.
3.The scavenging ability on super oxide anions generated by pyrogallol autoxidation system of lycopene and lutein was evaluated by means of spectrophotometry. The results showed that lycopene and lutein have obvious effect on scavenging super oxide anions and scavenging ability of lutein superior to lycopene.
4.The scavenging effects on hydroxyl radical of dioscoeaopposita thunb, apple, kiwifruit, orange, pear and tea were measured by 1, 10-phenanthroline-Fe2+-H2O2 and
methyl violet- Fe2+-H202 reaction system. The results showed scavenging ability of apple, kiwifruit, orange, pear decreased by subsequence. Methyl violet- Fe2+-H202 reaction system superior to 1,10-phenanthroline-Fe2+-H202 by comparing the hydroxyl radical scavenges effect of ascorbic acid and thiourea.
1．番茄红素的提取工艺研究 通过番茄红素提取过程中脱水方法、溶剂提取、纯化方法等各程序中实验参数的考察，确定了有效提取番茄红素的最佳工艺条件：首先用5g食盐对20g番茄果肉进行脱水，然后加丙酮处理，所得番茄用20ml乙酸乙酯提取，提取液浓缩后得到番茄红素油树脂，通过柱层析法纯化，获得的产品吸收光谱图与文献番茄红素的标准图谱相一致，本工艺可为番茄的深度开发提供方法参考和理论依据，具有推广应用价值。
2．叶黄素的提取工艺研究 通过叶黄素提取过程中各种实验因素的考察，确定了从万寿菊中提取叶黄素的最佳工艺：6g万寿菊粉通过正己烷萃取后浓缩再用3.3％的NaOH皂化处理，可以获得0.0795g叶黄素。并对其进行了薄层层析，此研究建立的工艺体系可为通过万寿菊获取叶黄素提供方法参考，从而为万寿菊的推广种植提供理论依据。
3．通过分光光度法研究了番茄红素和叶黄素对邻苯三酚自氧化产生超氧阴离子自由基的清除作用，结果表明这两种天然色素对超氧阴离子自由基均有明显的清除效果，相同浓度的叶黄素的清除率明显高于番茄红素。
4．利用甲基紫-Fe~(2+)-H_2O_2和Fe~(2+)-邻二氮菲-H_2O_2两种反应体系研究了天然产物猕猴桃、苹果、桔子、梨、红茶、绿茶和山药对·OH的清除能力，结果表明：苹果、猕猴桃、桔子、梨的清除·OH能力依次下降，红茶和绿茶清除能力相当。两种反应体系比较，甲基紫-Fe~(2+)-H_2O_2测定灵敏度高于Fe~(2+)-邻二氮菲-H_2O_2体系。
With the increasing improvement of living standard, people become to pursuit dietary nature and health. As a kind of functional food, Lycopene and lutein are widely studied because of their usage of preventing cancer and anti-cancer. In this paper, not only extraction methods and scavenging ability on super oxide anions of lycopene and lutein were studied, but also scavenging effect on hydroxyl radical of another natural products was measured. Detailed contents are followed:
1. Extraction of lycopene from fresh tomato was investigated in order to determine its dehydration methods, extraction solvent and purification technology. 5g salts were used in the dehydration of 20g tomato pulp. 20ml Ethyl acetate was used to extract lycopene from tomato tissue after being disposed with acetone. Through above extraction technology, 0.897mg lypocene can be obtained. Lypocene-containing oleoresin were gained after condense. Saponification and colummation layer analysis were used to sublimate lycopene oleoresin. Spectrogram of lycopene products gained is agreed with its standard Spectrogram. Colummation layer analysis superior to Saponification by comparing their extraction rate.
2.We investigate the technogical conditions for extracting lutein from marigold (Tagetes erecta) and determined its optimum technology. When concentration of NaOH is 3.3%, extracting time was two hours and extraction times was two times, 0. 0795g lutein products are obtained from 6g marigolds. It was analyzed by thin-layer chromatography on silica plates.
3.The scavenging ability on super oxide anions generated by pyrogallol autoxidation system of lycopene and lutein was evaluated by means of spectrophotometry. The results showed that lycopene and lutein have obvious effect on scavenging super oxide anions and scavenging ability of lutein superior to lycopene.
4.The scavenging effects on hydroxyl radical of dioscoeaopposita thunb, apple, kiwifruit, orange, pear and tea were measured by 1, 10-phenanthroline-Fe2+-H2O2 and
methyl violet- Fe2+-H202 reaction system. The results showed scavenging ability of apple, kiwifruit, orange, pear decreased by subsequence. Methyl violet- Fe2+-H202 reaction system superior to 1,10-phenanthroline-Fe2+-H202 by comparing the hydroxyl radical scavenges effect of ascorbic acid and thiourea.
引文
1. Nguyen M. L., Schwartz S. J. Food Technology, 1999, 53(2):38-45
2．孙庆杰，丁霄霖．食品与发酵工业，1997，23(4)：72-75
3．李增光，吴骥陶．食品与发酵工业，1991，16(2)：82-84
4. Crouzet J., Kanasawud P. Meth.Enzymol., 1992, 213:185-205
5．孙庆杰，丁霄霖．食品与发酵工业，1998，24(2)：47-51
6. Mascio P., Kaiser S., et al. Arch. Biochem. Biophys., 1989, 274:532-538
7. Miller N. J, Sampson J., Candeias L. P., et al. FEBS Lett., 1996, 384:240-242
8．赵文恩等．生物学杂志，2000，17(4)：4-6
9. Conn P.F., Lambert C., Land E. J., et al. Free Rad Res., 1992,16:401-408
10．赵文恩等．中国科学(B辑)，2002，32(4)：355-359
11. Franceschi S., Bidoli E. M., et al. Int J Cancer, 1994, 59:181-184
12. Burmey P. G., Comstock G. W., Morris J. S. Am J Clin. Nutr., 1989, 49:895-900
13. Kim D. J., Takasuka N., Kim J. M., et al. Cancer Lett., 1997, 120:15-22
14．孙庆杰，丁霄霖．中国食品添加剂，1998，(1)：1-6
15. Coodley C. O., et al. J. Womens Health, 1995, 4:303-311
16. Periquet B. A., et al. Aids, 1995, 9:887-893
17. Steven K. Clinton Nutrition Review, 1998, 56(2):35-51
18．张连富．农牧业产品，1998,8：18-21
19．刘心绮．食品资讯，1999，2：38-41
20. Stahl W., Sies H. Arch Biochemical, 1996, 336:1-9
21. Giorannucci E. epidemiologic literature, 1999, 91(4):317-331
22. Mirhthy L. N., et al. Food Technology, 1999, 53(2):38-45
23. Shanna S.K., et al. Italian J of Food.Science, 1996, 8(2):107-113
24．祝曙华．农牧产品开发，2001，5：14—15
25．周静等．新疆农垦科技，1992，4：25—29
26. Yen G. C., et al. Journal of the Chinese Agricultural Chemical Society, 1985, 19(3): 227-235
27．EP 0252501 AZ(1988)．(欧洲专利)
28．凌关庭等编．食品添加剂手册，北京：化学工业出版社，1989，243-244
29．蔡俊，邱雁临，谈小兰等．食品与发酵工业，2000，26(2)：50-53
30. Dubodel N. E, et al. Pischchevaya Promyshlennost, Moscow, 1995, 1:16
31．JP 7924940(1979)．(日本专利)
32．李玉环，王锋等．食品科学，2002，23(8)：146—150
33．岑宁，王杰，谢继志．江苏农学院学报，1996，17(2)：67-69
34. Gavrilov A.S. et al. Applied Biochemistry and Microbiology, 1996, 32(5): 492-494
35. Minura Y., et al. Biotechnology and Bioengineering, 1998, 58(2):306-308
36．张卫强，邓宇．食品工业科技，2002，23(5)：6-37
37．孙庆杰，丁霄霖．食品与发酵工业，1998，24(1)：3—6
38．平田真树．食品之开发，1995，31(3)：9—12
39. Stahl W., Sies H. L. BioPhysics, 1996, 336:1-9
40．孙庆杰，丁霄霖．食品与发酵工业，1997，23(4)：72-73
41．成坚，曾庆孝．食品与发酵工业，2000，26(2)：75—76
42. Hulten K. Cancer Control, 2001, 12:529-537
43. Toniolo P. A. J. Epidemiol, 2001,153:114-247
44. Hart D. J., Scott K. J. Food Chem., 1995, 54:101-111
45. Dwyer J.H.,Navab M. Circulate, 2001, 103:2922-2927
46. landrum J.T. Exp.Eye Res., 1997,65: 57-62.
47. Hammond B. R., Wooten B. R., Snodderly D. M. Optom Vis. Sci., 1997, 77: 499- 504
48. Mortensen A., Skibsted L. H., Sampson J., Rice～Evans C., Everett S. A. FEBS Letters, 1997, 418:91-97
49. Landrum A., et al. Ady Pharml, 1997,38:537-556
50. Berendschot T.T.,Goldbohm R. A., Klopping W. A., et al. Investigative Ophthalmology and Visual Science, 2000, 41:3322-3326.
51. Witzfum J.L., Steinberg D. J. Clin.Invest., 1991, 88:1785-1792
52. Wart F.G.,et al. Intl. J. Epidemid, 2001, 30:2136-2143
53. Howard A. N., et al. Int J Vitam Nutr. Res., 1996, 66:113-118
54. Jean S. P. J. Nutri., 1998, 128(10):1650-1656.
55. Bone R.A., et al. Exp Eye Res., 1997, 64:211
56. Snodderiy D. M., et al. Invest Ophthalmol Vis. Sci., 1991, 32:268
57. Hammond B. R., et al. Vision Res., 1996, 36:2001
58. Sharpe L.T., et al. Visions, 1998, 38:3233
59. Bone R. A., et al. Invest Ophthalmol Vis. Sci., 1988, 29:834
60. Hammond B. R., et al. J. OptsocAmA, 1997, 14:1187
61. Handelman G. J., et al. Invest Ophthalmol Vis Sci., 1989, 29:850
62．何泽超，章杰，赵晓琼．化工设计，2003，13(4)：10-12
63．盛爱武，陈翠云，谢应毅．仲恺农业技术学院学报，2001，14(4)：38-41
64．方允中，李文杰．自由基与酶．科学出版社，1989,272
65. Mccord J. M. Science, 1974, 185:529-531
66. Halliwell B., Cuteridge J.M.C. Oxford University Press, New York, 1985,218
67．陈瑗，周玫．自由基医学．人民军医出版社．北京，1991,231
68. Bulkley G. B. Surgery, 1983, 94:407
69. Sagone A. L., Greenwald J. J., Blanchine E. J. Lab Clin. Med., 1983, 101:97
70. Husain S. R., Cillard J., Cillard P. Phytochemistry, 1987, 26:2489
71. Havsteen B. Biochem. Pharm., 1983, 32:1141
72. Melzig M. E Planta Med., 1996, 62:20-21
73. Marbett K. J. Carcinogenesis, 1987, 8:1365
74. Stard A. A. Carcinogenesis, 1990, 11:529
75. Whorton A. R. J. Clin. Invest, 1985, 76:295
76. Ager A., Gordon J. L. J.Exp. Med., 1984, 159:592
77. Frederick V. L. Clin. Chem., 1993, 65:373
78. Fridovich I. Curr. Eye Res., 1984, 3:1
79. Mccord T. M., Fridobich I. J Biochem., 1968, 243:5753
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81．贾菊芳，数明章，胡本容等．营养学报，1994，23(1)：62—65
82．张英，唐莉莉，丁霄霖．营养学报，1998，20(3)：367-371
83．任继刚，刘沛霖，赵保路等．同济医科大学学报，1997，26(2)：112—115
84．郑学钦，胡春．中国药学杂志，1997，32(3)：140-142
85．宋俊梅，丁霄霖．食品科学，1997，18(9)：61—64
86．许申鸿，杭瑚．食品科学，1999，12：28-30
87．项光亚，杨瑜，阮金兰等．同济医科大学学报，2001，30(3)：211—213
88．黄沛力，曾昭晖．中国药学杂志，1996，35(3)：274-276
89．何纯莲，雷丽红，凌晓．光谱实验室，2003，20(1)：102-104
90．王春雨，牛淑敏，刘方．食品工业科技，2001，22(6)：21—22
2．孙庆杰，丁霄霖．食品与发酵工业，1997，23(4)：72-75
3．李增光，吴骥陶．食品与发酵工业，1991，16(2)：82-84
4. Crouzet J., Kanasawud P. Meth.Enzymol., 1992, 213:185-205
5．孙庆杰，丁霄霖．食品与发酵工业，1998，24(2)：47-51
6. Mascio P., Kaiser S., et al. Arch. Biochem. Biophys., 1989, 274:532-538
7. Miller N. J, Sampson J., Candeias L. P., et al. FEBS Lett., 1996, 384:240-242
8．赵文恩等．生物学杂志，2000，17(4)：4-6
9. Conn P.F., Lambert C., Land E. J., et al. Free Rad Res., 1992,16:401-408
10．赵文恩等．中国科学(B辑)，2002，32(4)：355-359
11. Franceschi S., Bidoli E. M., et al. Int J Cancer, 1994, 59:181-184
12. Burmey P. G., Comstock G. W., Morris J. S. Am J Clin. Nutr., 1989, 49:895-900
13. Kim D. J., Takasuka N., Kim J. M., et al. Cancer Lett., 1997, 120:15-22
14．孙庆杰，丁霄霖．中国食品添加剂，1998，(1)：1-6
15. Coodley C. O., et al. J. Womens Health, 1995, 4:303-311
16. Periquet B. A., et al. Aids, 1995, 9:887-893
17. Steven K. Clinton Nutrition Review, 1998, 56(2):35-51
18．张连富．农牧业产品，1998,8：18-21
19．刘心绮．食品资讯，1999，2：38-41
20. Stahl W., Sies H. Arch Biochemical, 1996, 336:1-9
21. Giorannucci E. epidemiologic literature, 1999, 91(4):317-331
22. Mirhthy L. N., et al. Food Technology, 1999, 53(2):38-45
23. Shanna S.K., et al. Italian J of Food.Science, 1996, 8(2):107-113
24．祝曙华．农牧产品开发，2001，5：14—15
25．周静等．新疆农垦科技，1992，4：25—29
26. Yen G. C., et al. Journal of the Chinese Agricultural Chemical Society, 1985, 19(3): 227-235
27．EP 0252501 AZ(1988)．(欧洲专利)
28．凌关庭等编．食品添加剂手册，北京：化学工业出版社，1989，243-244
29．蔡俊，邱雁临，谈小兰等．食品与发酵工业，2000，26(2)：50-53
30. Dubodel N. E, et al. Pischchevaya Promyshlennost, Moscow, 1995, 1:16
31．JP 7924940(1979)．(日本专利)
32．李玉环，王锋等．食品科学，2002，23(8)：146—150
33．岑宁，王杰，谢继志．江苏农学院学报，1996，17(2)：67-69
34. Gavrilov A.S. et al. Applied Biochemistry and Microbiology, 1996, 32(5): 492-494
35. Minura Y., et al. Biotechnology and Bioengineering, 1998, 58(2):306-308
36．张卫强，邓宇．食品工业科技，2002，23(5)：6-37
37．孙庆杰，丁霄霖．食品与发酵工业，1998，24(1)：3—6
38．平田真树．食品之开发，1995，31(3)：9—12
39. Stahl W., Sies H. L. BioPhysics, 1996, 336:1-9
40．孙庆杰，丁霄霖．食品与发酵工业，1997，23(4)：72-73
41．成坚，曾庆孝．食品与发酵工业，2000，26(2)：75—76
42. Hulten K. Cancer Control, 2001, 12:529-537
43. Toniolo P. A. J. Epidemiol, 2001,153:114-247
44. Hart D. J., Scott K. J. Food Chem., 1995, 54:101-111
45. Dwyer J.H.,Navab M. Circulate, 2001, 103:2922-2927
46. landrum J.T. Exp.Eye Res., 1997,65: 57-62.
47. Hammond B. R., Wooten B. R., Snodderly D. M. Optom Vis. Sci., 1997, 77: 499- 504
48. Mortensen A., Skibsted L. H., Sampson J., Rice～Evans C., Everett S. A. FEBS Letters, 1997, 418:91-97
49. Landrum A., et al. Ady Pharml, 1997,38:537-556
50. Berendschot T.T.,Goldbohm R. A., Klopping W. A., et al. Investigative Ophthalmology and Visual Science, 2000, 41:3322-3326.
51. Witzfum J.L., Steinberg D. J. Clin.Invest., 1991, 88:1785-1792
52. Wart F.G.,et al. Intl. J. Epidemid, 2001, 30:2136-2143
53. Howard A. N., et al. Int J Vitam Nutr. Res., 1996, 66:113-118
54. Jean S. P. J. Nutri., 1998, 128(10):1650-1656.
55. Bone R.A., et al. Exp Eye Res., 1997, 64:211
56. Snodderiy D. M., et al. Invest Ophthalmol Vis. Sci., 1991, 32:268
57. Hammond B. R., et al. Vision Res., 1996, 36:2001
58. Sharpe L.T., et al. Visions, 1998, 38:3233
59. Bone R. A., et al. Invest Ophthalmol Vis. Sci., 1988, 29:834
60. Hammond B. R., et al. J. OptsocAmA, 1997, 14:1187
61. Handelman G. J., et al. Invest Ophthalmol Vis Sci., 1989, 29:850
62．何泽超，章杰，赵晓琼．化工设计，2003，13(4)：10-12
63．盛爱武，陈翠云，谢应毅．仲恺农业技术学院学报，2001，14(4)：38-41
64．方允中，李文杰．自由基与酶．科学出版社，1989,272
65. Mccord J. M. Science, 1974, 185:529-531
66. Halliwell B., Cuteridge J.M.C. Oxford University Press, New York, 1985,218
67．陈瑗，周玫．自由基医学．人民军医出版社．北京，1991,231
68. Bulkley G. B. Surgery, 1983, 94:407
69. Sagone A. L., Greenwald J. J., Blanchine E. J. Lab Clin. Med., 1983, 101:97
70. Husain S. R., Cillard J., Cillard P. Phytochemistry, 1987, 26:2489
71. Havsteen B. Biochem. Pharm., 1983, 32:1141
72. Melzig M. E Planta Med., 1996, 62:20-21
73. Marbett K. J. Carcinogenesis, 1987, 8:1365
74. Stard A. A. Carcinogenesis, 1990, 11:529
75. Whorton A. R. J. Clin. Invest, 1985, 76:295
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