天然类胡萝卜素（叶黄素）的分离纯化及分子修饰
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摘要
本课题探索出一种简易分光光度法测定万寿菊中叶黄素及浸膏中叶黄素的含量,与其他现有方法(HPLC,AOAC)相比,具有操作快速简便(单样检测时间大约缩短1/3—1/2)、溶剂耗量小、检测成本低、环境污染小(节省溶剂约1/2)、精确度高的特点,适用于万寿菊及其粗、精加工产品中叶黄素的检测。
本课题以万寿菊提取浸膏为原料,设计出一种分离纯化工艺,得到高纯度叶黄素。最后得到的产品经过红外、紫外、核磁氢谱、核磁碳谱确定为高纯度的叶黄素,并且通过HPLC定性和定量检测,产品的纯度达到80%以上,产率能够达到70%以上。且该叶黄素分离纯化工艺适于在工业生产中实现。
本课题还设计了叶黄素向玉米黄素转化的工艺,得到最佳转化反应条件,且得到较高的转化率(从原来的5%提高到近40%)。工艺简单,易于应用于生产,产业化推广。
本课题还在玉米黄素分子修饰制得生物效价极高的虾青素分子方面做了探索。从而增加了虾青素的一种新来源,而且这是一种由天然色素分子化合物经过分子修饰制取的来源,克服了国内外文献报道的几种来源中存在的过程复杂、成本高的缺点,适于工业化的实现,有很好的应用前景。
The thesis mainly relates to a process to obtain xanthophylls concentrates of high purity from plant extracts. After drying under high vaccum, the crystalline is analyzed to be lutein with purity of more than 80 percent with IR, UV,NMR and HPLC.The thesis also relates to a process leading to the isomerization of Lutein to zeaxanthin and/or zeaxanthin-like epimers. The marigold extract is admixed with glycols and enough alkali. At the inert atmosphere, the mixture is heated to the temperature of about 100℃ for a period of time sufficient enough to complete the isomerization reaction. And the final substrate has a zeaxanthin content of about 40%, comparing to the initial content of 4.5%.A method to preparing .beta.-carotene derivatives such as canthaxanthin and astaxanthin is described. The method employs an in situ system to generate hypobromous acid as the oxidizing agent using a salt of sulfite, hydrogen sulfite orbisulfite in combination with a bromate salt. Astaxanthin and canthaxanthin are obtained in good yield with a significantly reduced reaction time.
本课题以万寿菊提取浸膏为原料,设计出一种分离纯化工艺,得到高纯度叶黄素。最后得到的产品经过红外、紫外、核磁氢谱、核磁碳谱确定为高纯度的叶黄素,并且通过HPLC定性和定量检测,产品的纯度达到80%以上,产率能够达到70%以上。且该叶黄素分离纯化工艺适于在工业生产中实现。
本课题还设计了叶黄素向玉米黄素转化的工艺,得到最佳转化反应条件,且得到较高的转化率(从原来的5%提高到近40%)。工艺简单,易于应用于生产,产业化推广。
本课题还在玉米黄素分子修饰制得生物效价极高的虾青素分子方面做了探索。从而增加了虾青素的一种新来源,而且这是一种由天然色素分子化合物经过分子修饰制取的来源,克服了国内外文献报道的几种来源中存在的过程复杂、成本高的缺点,适于工业化的实现,有很好的应用前景。
The thesis mainly relates to a process to obtain xanthophylls concentrates of high purity from plant extracts. After drying under high vaccum, the crystalline is analyzed to be lutein with purity of more than 80 percent with IR, UV,NMR and HPLC.The thesis also relates to a process leading to the isomerization of Lutein to zeaxanthin and/or zeaxanthin-like epimers. The marigold extract is admixed with glycols and enough alkali. At the inert atmosphere, the mixture is heated to the temperature of about 100℃ for a period of time sufficient enough to complete the isomerization reaction. And the final substrate has a zeaxanthin content of about 40%, comparing to the initial content of 4.5%.A method to preparing .beta.-carotene derivatives such as canthaxanthin and astaxanthin is described. The method employs an in situ system to generate hypobromous acid as the oxidizing agent using a salt of sulfite, hydrogen sulfite orbisulfite in combination with a bromate salt. Astaxanthin and canthaxanthin are obtained in good yield with a significantly reduced reaction time.
引文
1.王业勤,李勤生。天然类胡萝卜素研究进展、生产、应用。中国医药科技出版社。1997年。
2.王永华,梁世中.类胡萝卜素的结构和生理功能研究.2000,16,1-4.
3. Frederick Khachik; Gary R. Beecher; Noel F. Whittaker. Separation, identification and quantification of the major carotenoid and chlorophyll constituents in extracts of several green vegetables by Liquid Chromatography. J.Agri.Food Chem., 1986, 34, 603-616.
4. Anthony T D. Fat Soluble Vitamins, Their Biochemistry and Application. Trowbridgc: Redwood Bum Ltd., 1985, 1-6
5. Bendiah A.Beta-carotene and the immune response. Proc Nutr Soc, 1991, 50:263—274
6. Sies H Witheim, Vitamin E and C, beta-carotene, and other carotenoids as antioxidants. Am J clin Nutr, 1995, 62:1315—1321
7. Jozsef Deli, Zoltan Matus. Carotenoid composition in the fruits of asparagus officinalis. J.Agri.Food Chem., 2000, 48, 2793-2796.
8.赵文恩,韩雅珊等.类胡萝卜素对H_2O_2—NaOCL体系生成的~1O_2的淬灭作用.生物物理学报,1997,13(1):137—142
9. Gayle K.Gregory, Tung-Shan Chen. Quantitative analysis of lutein esters in marigold flowers(tagetes erecta) by high performance liquid chromatogyaphy; J.Food Sci., 1986, 51(4), 1093-1095.
10. Gerster H. Potential role of beta-carotene in the prevention of cardiovascular disease. Internat J Vit Nutr Res, 1991, 612:277—291
11. DanielR.Kull; Hanspeter pfander.Isolation and structural elucidation of two (Z)-isomers of lutein from the petals of Rape(Brassica napus). J.Agri. Food Chem. 1997.45.4201-4203.
12. D.Homero-Mendes,M.I.Minguez-Mosquera.Carotenoid pigments in Rosa mosqueta hips,an alternative carotenoid source for foods. J.Agric.Food Chem.,2000,48,825-828.
13. W.Leith Hadden,Ruth H.Watkins. Carotenoid composition of marigold(Tagetes erecta) flower extract used as nutritional supplement. J.Agri.Food Chem., 1999,47,4189-4194.
14. Adriana Z Mercadante,Andrea Steck.Carotenoids from Guava (Psidium guajava L.): Isolation and Structure Elucidation. J. Agri.Food Chem.,1999,47,145-151.
15. Frederick Khachik, Gerhard Englert. Isolation and structural elucidation of the geometrical isomers of lutein and zeaxanthin in extracts from human plasma. J. Chromatogr., 582(1992) 153-166.
16. Curt Emenhiser,Nada Simunovic.Separation of geometrical carotenoid isomers in biological extracts using a polyemeric C30 column in reversed-phase liquid chromatography. J.Agri. Food Chem.,1996,44,3887-3893.
17. F.Khachik,Gary R. Beecher.Sepatation and identification of carotenoids and their oxidation products in the extracts of human plasma. Anal.Chem.,1992,64,2111-2122.
18. B.H.Chen , H. Yang. Characterization of major carotenoids in water convolvulus (Ipomoea aquatica) by open-column thin-layer and high-performance liquid chromatography J Chromatogr., 543(1991), 147-155.
19. C.Y.Tai,B.H.Chen. Analysis and stability of carotenoids in the flowers of daylily (Hemerocallis disticha) as affected by various treatments. J.Agri.Food Chem.,2000,48,5962-5968.
20. Bendick.A.Recent advances in clicnical research involving carotenoids. Pure Appl.Chem.,1994,66,1017-1024.
21. C.Y.Tai,B.H.Chen. Analysis and stability of carotenoids in the flowers of daylily (Hemerocallis disticha) as affected by various treatments. J.Agri.Food Chem.,2000,48,5962-5968.
22. B.H.Chen, H. Yang. Characterization of major carotenoids in water convolvulus (Ipomoea aquatiea) by open-column thin-layer and high-performance liquid chromatography.J Chromatogr., 543(1991), 147-155.
23. Abteilung fur Vitamin, Ernahrungsforachuug. Synthses von optisch aktiven, naturlichen carotinoiden and stukturell verwandten Naturprodukten Ⅳ Synthesis von(3R, 3'R, 6'R)-Lutein; Helv.Chin.Acta., 1980, 63, 1451-1455.
24. Breithaupt D.E, Sehwack W. Determination of free and bound carotenoids in paprika (Capsicum annuum L.)by LC/MS [J]. European Food Research and Technology. 2000, 211(1): 52—55
25. Norman I. Krinsky, Mary D. Russett, Garry J.; Structural and geometrical isomers of carotenoids in human plasma. J. Nutr., 120, 1654-1662.
26. Katherine S.Epler, Lane C.Sander. Evaluation of reversed phase liquid chromatographic column for recovery and selectivity of seleted carotenoids. J.Chromatogr., 595(1992) 89-101.
27. Adam M.Gilmore, Harry Y.Yamamaoto. Resolution of lutein and zeaxanthin using a non-endcapped, highly carbon-loaded C_(30) high performance liquid chromatographic column. J. Chromatogr., 43(1991), 137-145.
28.凌关庭。可供开发新型食品添加剂(Ⅲ)万寿菊色素及其生理功能粮食与油脂,2002年第12期。
29. Gayle K. Gregory, Tung-Shan Chen. Quantitative analysis of lutein esters in marigold flowers(tagetes erecta) by high performance liquid chromatogyaphy; J.Food Sci., 1986, 51(4), 1093-1095.
30. J.I.X Antony. Luteln[J]. The world of Food ingredients, 2001, 4—5: 65—67.
31. W.L.Hadden et al. Carotenoids in Marigold flower extract [J]. J. Agric Food chem. 1999, 47(10); 4189—4193.
32. D.Hornero-Mendes, M.I.Minguez-Mosquera.Xanthophyll esterification accompanying carotenoid overaccumulation in chromoplast of capsicum annuum ripening fruits is a constitutive process and useful for ripeness index; J. Agric. Food Chem., 2000, 48, 1617-1622.
33. Torrison O J, Christiansen R.Requirements for carotenoids in fish diets[J].J Appl Ichthyol, 1995, 11:225—230
34. Torrissen O J, R W Hardy, K D Shearer. Pigmentation of salmonids—carotenoid deposition and metabolism[J]. CRC Crit Rev Aquat Sci, 1989, (1): 209—225.
35. Lee SH, Min DB. Effects, quenching Mechanisms, and Ki-netics of Carotenoids in Chlorophyll-Sensitized Photooxidation of Soyean Oil[J]. Agri Fiid Chim, 1990, 35(38):1630—1634
36. Miki W.Biological functions and activitieis of animal carotenoids[J].Pure & Appl Chem, 1991(63): 141—146。
37. Jyonouchi H, Sun S, Gross MD. Astaxanthin, a carotenoid withiout vitamin a activity, augments antibody responses in cultures including T-helper cell clones and suboptimal doses of antigen[J].Nutr, 1995, 125(10): 2483
38. Woodall A.A., Brinon G, & Jackson M.J., Carotenoids and protection of phospholipids in solution or in liposomes against oxidation by peroxyl radicals. Biochim. Biophys. Acta., 1997, 1336(3):578—586
39. Nakagawa, Kang S.D., Park D.K., et al. Inhibition of β-carotene and astaxanthin of NADPH-dependent microsornal phospholipid peroxidation, J. Nutr. Sci, Vitaminol., Tokyo, 1997, 43(3): 345—355
40. Tanaka T, Makita H, Ohnishi H, et al. Chemoprevention of ratoral carcinogensis by naturally occurrring xanthophyll, astaxanthin and canthaxanthin in [J]. Cancer Res, 1995, 55(18): 4059—4064.
41. Nishino H., Cancer prevention by carotenoids.Mutat Res., 1998(402):159—163
42. Gradelet S., Lebon A.M., Berges R., et al., Dietary carotenoids inhibit aflatoxin Bl-induced liver preneoplastic foci and DNA damage in the rat.Carcinogenesis, 1998, Mar., 19(3):403—411
43. Savoure X., Briand G., Amory T. M. C., et al., Vitamin A status and metabolism of cutaneous poly amines in the bairless mouse after UV irradiation: action of beta-carotene and astaxanthin. Int. J. Vitam-Nutr. Res., 1995, 65(2):79—86
44. Jyonouchi H., Zhang L., Gross M., et al., Immunomodulating actions of carorenoids; enhancement of in vivo and invitro antibody production to T-dependent antigenris. Nuti, Cancer, 1994, 21(1):47
45. Grung M. etal. Algal carotenoids. Secondary carotenoids Z. H. P. aplanospores as a source of(3s, 3's)-astaxanthin esters[J].J.Appl.Phycol, 1992, 4(2): 165—171
46. Sigurgisladottirs et al. Eflects of feeding natural tocopherols and astaxanthin or Atlantic salmon (Salinosalar) fillet quality [J]. Food Res Int. 1994, 27(1): 23—32
47. Andrewes A. G. Phaff H.J. Phytochemistry, 1976, 15(1): 1003—1007
48.戴亦军,秦怀兰,袁生。红发夫酵母(Phaffia rhodozyma)及其虾青素的研究开发[J].生物技术,1996,6(3):41—4。
49.徐学明,金征宇,刘当慧 饲用色素虾青素的生产和应用[J]。饲料工业,2000,21(2):12—14。
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2.王永华,梁世中.类胡萝卜素的结构和生理功能研究.2000,16,1-4.
3. Frederick Khachik; Gary R. Beecher; Noel F. Whittaker. Separation, identification and quantification of the major carotenoid and chlorophyll constituents in extracts of several green vegetables by Liquid Chromatography. J.Agri.Food Chem., 1986, 34, 603-616.
4. Anthony T D. Fat Soluble Vitamins, Their Biochemistry and Application. Trowbridgc: Redwood Bum Ltd., 1985, 1-6
5. Bendiah A.Beta-carotene and the immune response. Proc Nutr Soc, 1991, 50:263—274
6. Sies H Witheim, Vitamin E and C, beta-carotene, and other carotenoids as antioxidants. Am J clin Nutr, 1995, 62:1315—1321
7. Jozsef Deli, Zoltan Matus. Carotenoid composition in the fruits of asparagus officinalis. J.Agri.Food Chem., 2000, 48, 2793-2796.
8.赵文恩,韩雅珊等.类胡萝卜素对H_2O_2—NaOCL体系生成的~1O_2的淬灭作用.生物物理学报,1997,13(1):137—142
9. Gayle K.Gregory, Tung-Shan Chen. Quantitative analysis of lutein esters in marigold flowers(tagetes erecta) by high performance liquid chromatogyaphy; J.Food Sci., 1986, 51(4), 1093-1095.
10. Gerster H. Potential role of beta-carotene in the prevention of cardiovascular disease. Internat J Vit Nutr Res, 1991, 612:277—291
11. DanielR.Kull; Hanspeter pfander.Isolation and structural elucidation of two (Z)-isomers of lutein from the petals of Rape(Brassica napus). J.Agri. Food Chem. 1997.45.4201-4203.
12. D.Homero-Mendes,M.I.Minguez-Mosquera.Carotenoid pigments in Rosa mosqueta hips,an alternative carotenoid source for foods. J.Agric.Food Chem.,2000,48,825-828.
13. W.Leith Hadden,Ruth H.Watkins. Carotenoid composition of marigold(Tagetes erecta) flower extract used as nutritional supplement. J.Agri.Food Chem., 1999,47,4189-4194.
14. Adriana Z Mercadante,Andrea Steck.Carotenoids from Guava (Psidium guajava L.): Isolation and Structure Elucidation. J. Agri.Food Chem.,1999,47,145-151.
15. Frederick Khachik, Gerhard Englert. Isolation and structural elucidation of the geometrical isomers of lutein and zeaxanthin in extracts from human plasma. J. Chromatogr., 582(1992) 153-166.
16. Curt Emenhiser,Nada Simunovic.Separation of geometrical carotenoid isomers in biological extracts using a polyemeric C30 column in reversed-phase liquid chromatography. J.Agri. Food Chem.,1996,44,3887-3893.
17. F.Khachik,Gary R. Beecher.Sepatation and identification of carotenoids and their oxidation products in the extracts of human plasma. Anal.Chem.,1992,64,2111-2122.
18. B.H.Chen , H. Yang. Characterization of major carotenoids in water convolvulus (Ipomoea aquatica) by open-column thin-layer and high-performance liquid chromatography J Chromatogr., 543(1991), 147-155.
19. C.Y.Tai,B.H.Chen. Analysis and stability of carotenoids in the flowers of daylily (Hemerocallis disticha) as affected by various treatments. J.Agri.Food Chem.,2000,48,5962-5968.
20. Bendick.A.Recent advances in clicnical research involving carotenoids. Pure Appl.Chem.,1994,66,1017-1024.
21. C.Y.Tai,B.H.Chen. Analysis and stability of carotenoids in the flowers of daylily (Hemerocallis disticha) as affected by various treatments. J.Agri.Food Chem.,2000,48,5962-5968.
22. B.H.Chen, H. Yang. Characterization of major carotenoids in water convolvulus (Ipomoea aquatiea) by open-column thin-layer and high-performance liquid chromatography.J Chromatogr., 543(1991), 147-155.
23. Abteilung fur Vitamin, Ernahrungsforachuug. Synthses von optisch aktiven, naturlichen carotinoiden and stukturell verwandten Naturprodukten Ⅳ Synthesis von(3R, 3'R, 6'R)-Lutein; Helv.Chin.Acta., 1980, 63, 1451-1455.
24. Breithaupt D.E, Sehwack W. Determination of free and bound carotenoids in paprika (Capsicum annuum L.)by LC/MS [J]. European Food Research and Technology. 2000, 211(1): 52—55
25. Norman I. Krinsky, Mary D. Russett, Garry J.; Structural and geometrical isomers of carotenoids in human plasma. J. Nutr., 120, 1654-1662.
26. Katherine S.Epler, Lane C.Sander. Evaluation of reversed phase liquid chromatographic column for recovery and selectivity of seleted carotenoids. J.Chromatogr., 595(1992) 89-101.
27. Adam M.Gilmore, Harry Y.Yamamaoto. Resolution of lutein and zeaxanthin using a non-endcapped, highly carbon-loaded C_(30) high performance liquid chromatographic column. J. Chromatogr., 43(1991), 137-145.
28.凌关庭。可供开发新型食品添加剂(Ⅲ)万寿菊色素及其生理功能粮食与油脂,2002年第12期。
29. Gayle K. Gregory, Tung-Shan Chen. Quantitative analysis of lutein esters in marigold flowers(tagetes erecta) by high performance liquid chromatogyaphy; J.Food Sci., 1986, 51(4), 1093-1095.
30. J.I.X Antony. Luteln[J]. The world of Food ingredients, 2001, 4—5: 65—67.
31. W.L.Hadden et al. Carotenoids in Marigold flower extract [J]. J. Agric Food chem. 1999, 47(10); 4189—4193.
32. D.Hornero-Mendes, M.I.Minguez-Mosquera.Xanthophyll esterification accompanying carotenoid overaccumulation in chromoplast of capsicum annuum ripening fruits is a constitutive process and useful for ripeness index; J. Agric. Food Chem., 2000, 48, 1617-1622.
33. Torrison O J, Christiansen R.Requirements for carotenoids in fish diets[J].J Appl Ichthyol, 1995, 11:225—230
34. Torrissen O J, R W Hardy, K D Shearer. Pigmentation of salmonids—carotenoid deposition and metabolism[J]. CRC Crit Rev Aquat Sci, 1989, (1): 209—225.
35. Lee SH, Min DB. Effects, quenching Mechanisms, and Ki-netics of Carotenoids in Chlorophyll-Sensitized Photooxidation of Soyean Oil[J]. Agri Fiid Chim, 1990, 35(38):1630—1634
36. Miki W.Biological functions and activitieis of animal carotenoids[J].Pure & Appl Chem, 1991(63): 141—146。
37. Jyonouchi H, Sun S, Gross MD. Astaxanthin, a carotenoid withiout vitamin a activity, augments antibody responses in cultures including T-helper cell clones and suboptimal doses of antigen[J].Nutr, 1995, 125(10): 2483
38. Woodall A.A., Brinon G, & Jackson M.J., Carotenoids and protection of phospholipids in solution or in liposomes against oxidation by peroxyl radicals. Biochim. Biophys. Acta., 1997, 1336(3):578—586
39. Nakagawa, Kang S.D., Park D.K., et al. Inhibition of β-carotene and astaxanthin of NADPH-dependent microsornal phospholipid peroxidation, J. Nutr. Sci, Vitaminol., Tokyo, 1997, 43(3): 345—355
40. Tanaka T, Makita H, Ohnishi H, et al. Chemoprevention of ratoral carcinogensis by naturally occurrring xanthophyll, astaxanthin and canthaxanthin in [J]. Cancer Res, 1995, 55(18): 4059—4064.
41. Nishino H., Cancer prevention by carotenoids.Mutat Res., 1998(402):159—163
42. Gradelet S., Lebon A.M., Berges R., et al., Dietary carotenoids inhibit aflatoxin Bl-induced liver preneoplastic foci and DNA damage in the rat.Carcinogenesis, 1998, Mar., 19(3):403—411
43. Savoure X., Briand G., Amory T. M. C., et al., Vitamin A status and metabolism of cutaneous poly amines in the bairless mouse after UV irradiation: action of beta-carotene and astaxanthin. Int. J. Vitam-Nutr. Res., 1995, 65(2):79—86
44. Jyonouchi H., Zhang L., Gross M., et al., Immunomodulating actions of carorenoids; enhancement of in vivo and invitro antibody production to T-dependent antigenris. Nuti, Cancer, 1994, 21(1):47
45. Grung M. etal. Algal carotenoids. Secondary carotenoids Z. H. P. aplanospores as a source of(3s, 3's)-astaxanthin esters[J].J.Appl.Phycol, 1992, 4(2): 165—171
46. Sigurgisladottirs et al. Eflects of feeding natural tocopherols and astaxanthin or Atlantic salmon (Salinosalar) fillet quality [J]. Food Res Int. 1994, 27(1): 23—32
47. Andrewes A. G. Phaff H.J. Phytochemistry, 1976, 15(1): 1003—1007
48.戴亦军,秦怀兰,袁生。红发夫酵母(Phaffia rhodozyma)及其虾青素的研究开发[J].生物技术,1996,6(3):41—4。
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