羊脾脏多肽的酶法制备及体外抗氧化机理的研究
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摘要
本研究以羊脾脏蛋白为原料，用木瓜蛋白酶和胰蛋白酶双酶水解，选择最佳水解条件。结果表明：温度60℃C，pH7.0，加酶量5.0％，时间5.5h为最佳水解条件，水解率达60.59％，二苯代苦味酰基自由基(DPPH)值即抗氧化活性中等偏上。用Scphadex G—25分离水解混合物，分别收集得到四个峰，其产物编号为F_1、F_2、F_3、F_4，并进行DPPH测试以确定其抗氧化活性，测得F_3抗氧化能力较强。对F_3进行Tricine—SDS—PAGE电泳分析，测定其分子量。
通过对F_3进行非脂质体系和脂质体系体外抗氧化活性的测试表明：在几种不同的自由基产生体系中：F_3能显著抑制·OH的生成，随浓度的增大，F_3清除DPPH能力增强；并且抗氧化能力随浓度的增加而增强；F_3对O_2~(?)没有明显的清除作用。采用DPPH法对复合抗氧化剂的活性进行评价：柠檬酸+脾肽的清除能力比单独的脾肽和单独的柠檬酸效果都显著(P＜0.01)，但抗坏血酸与脾肽的复合抗氧化效果不显著。在卵磷脂制备的脂质体模拟细胞膜上的抗氧化体系和猪油体系中，F_3均表现出一定的抗氧化活性，并随添加浓度的增加，其抗氧化活性不断增强。
In the present research papain and trypsin were used to hydrolyze spleen protein and then the antioxidant properties of the hydrolysis were analyzed. It was done to abtain the optimal condition of hydrolysis of spleen protein. The optimum hydrolysis parameters were: the enzyme application accounted for 5.0%(enzyme/substrate),reaction conditions of pH 7.0, temperature 60#, time 5.5 hours and the degree of hydrolysis being 60.59% with stronger antioxidative activities. The hydrolysate was fractionated by gel filtration on a Sephadex G-25 column.Four fractions(their serial number is F1, F2, F3, F4) were gained,and then their antioxidation activity were tested by DPPH . The result showed that F3 was the most active antioxidant compared with the other fractions .The fraction was analyzed by Tricine-SDS-PAGE ans the molecular weight was estimated by this method.
Experiments about the inhibitory effect of the peptides FS on lipoxygenase, the auto-oxidizing of lipid were performed . It turned out : In different free radical generating systems: F3 was able to inhibit the OH effectively with the larger the concentration; Scavenging effects on 1,1-Diphenyl-2-picryl-hydrazyl(DPPH), the antioxidative activities of F3 were stronger with the increasing concentrations; However, FS wasnot effectively eliminated O2-. A Significant(p < 0.01) synergistic effects of citric acid plus spleen polypeptides were obtained by DPPH method .however no significant synergistic effects when employing assorbic acid plus spleen polypeptides; In the systems of swine
lard and phosphatidylcholine liposome, it was found Fa had some extend of antioxidant activities, the larger the concentration, the stronger the antiooxidation activity.
通过对F_3进行非脂质体系和脂质体系体外抗氧化活性的测试表明：在几种不同的自由基产生体系中：F_3能显著抑制·OH的生成，随浓度的增大，F_3清除DPPH能力增强；并且抗氧化能力随浓度的增加而增强；F_3对O_2~(?)没有明显的清除作用。采用DPPH法对复合抗氧化剂的活性进行评价：柠檬酸+脾肽的清除能力比单独的脾肽和单独的柠檬酸效果都显著(P＜0.01)，但抗坏血酸与脾肽的复合抗氧化效果不显著。在卵磷脂制备的脂质体模拟细胞膜上的抗氧化体系和猪油体系中，F_3均表现出一定的抗氧化活性，并随添加浓度的增加，其抗氧化活性不断增强。
In the present research papain and trypsin were used to hydrolyze spleen protein and then the antioxidant properties of the hydrolysis were analyzed. It was done to abtain the optimal condition of hydrolysis of spleen protein. The optimum hydrolysis parameters were: the enzyme application accounted for 5.0%(enzyme/substrate),reaction conditions of pH 7.0, temperature 60#, time 5.5 hours and the degree of hydrolysis being 60.59% with stronger antioxidative activities. The hydrolysate was fractionated by gel filtration on a Sephadex G-25 column.Four fractions(their serial number is F1, F2, F3, F4) were gained,and then their antioxidation activity were tested by DPPH . The result showed that F3 was the most active antioxidant compared with the other fractions .The fraction was analyzed by Tricine-SDS-PAGE ans the molecular weight was estimated by this method.
Experiments about the inhibitory effect of the peptides FS on lipoxygenase, the auto-oxidizing of lipid were performed . It turned out : In different free radical generating systems: F3 was able to inhibit the OH effectively with the larger the concentration; Scavenging effects on 1,1-Diphenyl-2-picryl-hydrazyl(DPPH), the antioxidative activities of F3 were stronger with the increasing concentrations; However, FS wasnot effectively eliminated O2-. A Significant(p < 0.01) synergistic effects of citric acid plus spleen polypeptides were obtained by DPPH method .however no significant synergistic effects when employing assorbic acid plus spleen polypeptides; In the systems of swine
lard and phosphatidylcholine liposome, it was found Fa had some extend of antioxidant activities, the larger the concentration, the stronger the antiooxidation activity.
引文
1．翁新楚．郑州粮食学院学报，1993(3)：20-29
2．方允中等．自由基与酶，北京：科学出版社，1989
3．徐学兵等．油脂化学，北京：中国商业出版社，1993
4．任国谱，佘兵，李顺录．蛋白质及其衍生物的抗氧化性能．中国油脂1997．22．(4)
5．凌关庭．食品抗氧化剂及其进展(Ⅰ)，粮食与油脂。2000．6
6．夏象东，品飞杰，抬速祥．抗氧化剂的功效及氧化剂活性的体外分析评价．食品科学，2001．22．(1)
7．杨祥，韦小英，阮征．国内外天然食品抗氧化剂的研究进展，食品科学，2002．23(10)
8．凌关庭．食品与油脂，2000，6：45—48
9．邹坤，张如意，赵玉英．天然产物抗氧化构效关系及作用机理的研究概况．天然产物研究与开发，1993，3，5(1)
10．抗氧化剂的抗氧化活性的测定方法及其评价．中国油脂，2000，25，(6)
11．万素英等．食品抗氧化剂，北京：中国轻工业出版社，1998
12．郑晶泉．抗氧化剂抗氧化实验研究进展．国外医学卫生学分册，2000，27(1)
13．赖颖珍，郭俊钦．禽肉肌肽之萃取及其抗氧化性．食品科学(台湾)，1999，26(1)，16—27
14．方允中等，自由基与酶，北京：科学出版社，1989
15．孟繁振．保尔佳．中国新药杂志．1996，5(6)：6
16．高贵波，李正平等．脾脏的提取及其治疗恶性肿瘤的临床辅助性疗效．中国康复．1999，14(3)：154—155
17．赵锐，顾谦群，管华诗．天然活性肽的研究进展．天然产物研究与开发，2000，12(4)：84—91
18．葛轶群，俞建瑛，宋肇文等．生物活性肽的研究进展．中国生化药物杂志，1998，19(6)：404—406
19．李虹奇．活性多肽蛋白质研究近况．中草药，1993，72(24)：373—378
20．周秋丽，王丽娟，郭颖法．鹿茸多肽药理作用研究．天然产物研究与开发，1998，10(3)：57—60
21．纪建国，叶蕴华．植物多肽类化合物研究进展．天然产物研究与开发，1998，10(3)：80—86
22．北京师范大学生物系．基础生物化学实验．高等教育出版社，1982
23．食品分析．中国轻工业出版社，1994
24．石继红，赵泳同，张英起等．应用SDS—PAGE显示小分子多肽技术的探讨．生物工程进展，2001，21(1)：38—40
25．魏安池，李喜红，吴新玲．天然抗氧化剂研究概况．郑州工程学院学报，2000，21(4)：62
—66
26．黄惠华，何铁剑，许南燕．木瓜蛋白酶对大豆分离蛋白的水解作用研究．食品工业，1999．3：29—31
27．陈文荣，邱业先．叶蛋白酶促水解物营养价值的研究。食品科学，2002，23(6)：100—102
28．郑琳，蒲训，毕玉蓉．白阿魏侧耳子实体抗氧化活性的研究．中国食用菌，2002 22(1)：23—25
29．王爱国，罗广华．羟自由基启动下的脱氧核糖降解及其产物的TBA反应．生物化学与生物物理进展，1993 20(2)：150—152
30．彭长连，陈少薇等．用清除有机自由基DPPH法评价植物抗氧化能力．生物化学与生物物理进展，2000，27(6)：658—661
31．祁可宗，王林安．自由基和生物抗氧化系统理论与外科学的关系．安徽农业大学学报，1996，23(2)：171—174
32. Manach C, Morand C, Texier O, et al. Quercetin metabolites in plas ma of rats fed diets containing rutin or quercetin [J].J Nutr, 1995,125:1911—1912
33. Holl man P C H, de Vires J H M, Van Lee Wen S D, et al . Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy Volunteers [J]. Am J Clin Nutr, 1995; 62:1276—1282
34. Sousc RL, et al. Arch. Biochem. Biophys, 1985: 240—345
35. T. Matsuno. Et al. Biochem. P HARMACOL, 1990,39(7):1255
36. Buckley, D.J.,et al. Influence of dietary vitamin E on the oxidative stability and quality of pigmeat. J. Anim. Sci, 1995, 73:122—130
37. Akamittath, J.G., et al. Lipid peroxidation and color stability in restructured meat systems during frozen storage. J. Food Sci, 1990, 55:1513—1517
38. T. Matsuno, et al. Biochem. Pharmacol. 1987, 36(10): 1613
39. S Beyeler, et al. Biochem, Pharmacol. 1988,37(10):1971
40. R .A .Larson .Phtochemistry ,1988 ,27 (4) :969
41. Decker, E A., et al. Inhibition of Lipid oxidation bycarnosine JAOCS, 1990,67: 650— 652
42. Maridonononneau-PariniI, et al . Pharm. Res Cpmmun, 1986, 18:61
43. MJ. Luughton, etal. Biochem. Pharmavol , 1989, 38 (17): 2859
44. Pryor, W, A, Kaufman, M, J, church, D. F, J. Drg. chem. 1985, 50 (2): 281-283
45. Halliwell B etal. chem. Toxicol, 1995, 33 (7): 601-617
46. Halliwell B and chirico S. Am J clin. Nutr, 1993, 57 (Suppl): 715-725
47. Larrauri J A, Sanchez Moreno C, Saura-Calixto F. Effect of temperature on the free
radical scavenging capacity of extracts from red and white grape pomace peels. J Agric Foo Chem, 1998, 46(7):2694-2697
48. Yokozawa T, Dong E, Natagawa T, et al. In vivo studies on the radical-scavenging activity of tea. J Agric Food Chem, 1998,46(6):2143-2150
49. Decker, E.A&Faraji, H. Imhibition of lipid by carnosine. JAOCS, 1990,67:650
50. Boldyrev, A.A.,Dupin, h.M.,Pindel, E.V.& Severin, S.E. Antioxidative properties of histidine-containing dipeptides from skeletal muscles of vertebrates. Comparative Biochemical. Physioligy, 1988,89B:245-250
51. Decker, E.A. & Crum, A.D. Inhibition of oxidative rancidity in salted ground pork by carnosine. J. Food Sci., 1991, 56:1179
52. Decker, E.A. & Crum, A.D. Antioxidant activity of carnosine in cooked ground pork, Mea Sci., 1993, 34:245
53. K M Chan, et al. Journal of Food Science , 1993, 58(1), 1—4
54. R.E. Hayes, G.N. Book Walter and E.B. Bagley. J. Food Sci.,1997,42(6)
55. Kisoon Rhee et al.J. Food Sci., 1979,44:1132
56. Hua—Ming then et al.,Structural analysis of antioxidative peptides from soybean β —conglyon. J. Agri. Food Chem.,1995,43:574—578
57. Hackler L. kastin A J. Zadina J E. Isolation of novel peptides with a unique binding profile from human brain cortex: Tyr_k_MIF_1. peptides. 1994, 15 (6): 945—950
58. Cheng D L Shao Y zhao K et al. Penpeptides from the roots of Aster tataricus. Pharmazie, 1996, 51 (1): 185—186
59. Yenhua He, Fereidoon Shahidi. Antioxidant activity of green tea and its catechins in a fish meat model system. J Agric Food Chem, 1997,45:4262-4265
60. Barry Halliwell et al. The Deroxyribose Method: A Simple of "Test=Tube"Assay For Determination of Rate Constants for Reactions Hydroxyl Radicals. Analytical Biochemistry. 1987,165:215=219
61. Kohen, R., et al. Antioxidant activity of carnosine, homocarno-sine, and anserine present in muscle and brain. Proc. Nat. Acad. Sci. USA, 1988,85:3175-3179
2．方允中等．自由基与酶，北京：科学出版社，1989
3．徐学兵等．油脂化学，北京：中国商业出版社，1993
4．任国谱，佘兵，李顺录．蛋白质及其衍生物的抗氧化性能．中国油脂1997．22．(4)
5．凌关庭．食品抗氧化剂及其进展(Ⅰ)，粮食与油脂。2000．6
6．夏象东，品飞杰，抬速祥．抗氧化剂的功效及氧化剂活性的体外分析评价．食品科学，2001．22．(1)
7．杨祥，韦小英，阮征．国内外天然食品抗氧化剂的研究进展，食品科学，2002．23(10)
8．凌关庭．食品与油脂，2000，6：45—48
9．邹坤，张如意，赵玉英．天然产物抗氧化构效关系及作用机理的研究概况．天然产物研究与开发，1993，3，5(1)
10．抗氧化剂的抗氧化活性的测定方法及其评价．中国油脂，2000，25，(6)
11．万素英等．食品抗氧化剂，北京：中国轻工业出版社，1998
12．郑晶泉．抗氧化剂抗氧化实验研究进展．国外医学卫生学分册，2000，27(1)
13．赖颖珍，郭俊钦．禽肉肌肽之萃取及其抗氧化性．食品科学(台湾)，1999，26(1)，16—27
14．方允中等，自由基与酶，北京：科学出版社，1989
15．孟繁振．保尔佳．中国新药杂志．1996，5(6)：6
16．高贵波，李正平等．脾脏的提取及其治疗恶性肿瘤的临床辅助性疗效．中国康复．1999，14(3)：154—155
17．赵锐，顾谦群，管华诗．天然活性肽的研究进展．天然产物研究与开发，2000，12(4)：84—91
18．葛轶群，俞建瑛，宋肇文等．生物活性肽的研究进展．中国生化药物杂志，1998，19(6)：404—406
19．李虹奇．活性多肽蛋白质研究近况．中草药，1993，72(24)：373—378
20．周秋丽，王丽娟，郭颖法．鹿茸多肽药理作用研究．天然产物研究与开发，1998，10(3)：57—60
21．纪建国，叶蕴华．植物多肽类化合物研究进展．天然产物研究与开发，1998，10(3)：80—86
22．北京师范大学生物系．基础生物化学实验．高等教育出版社，1982
23．食品分析．中国轻工业出版社，1994
24．石继红，赵泳同，张英起等．应用SDS—PAGE显示小分子多肽技术的探讨．生物工程进展，2001，21(1)：38—40
25．魏安池，李喜红，吴新玲．天然抗氧化剂研究概况．郑州工程学院学报，2000，21(4)：62
—66
26．黄惠华，何铁剑，许南燕．木瓜蛋白酶对大豆分离蛋白的水解作用研究．食品工业，1999．3：29—31
27．陈文荣，邱业先．叶蛋白酶促水解物营养价值的研究。食品科学，2002，23(6)：100—102
28．郑琳，蒲训，毕玉蓉．白阿魏侧耳子实体抗氧化活性的研究．中国食用菌，2002 22(1)：23—25
29．王爱国，罗广华．羟自由基启动下的脱氧核糖降解及其产物的TBA反应．生物化学与生物物理进展，1993 20(2)：150—152
30．彭长连，陈少薇等．用清除有机自由基DPPH法评价植物抗氧化能力．生物化学与生物物理进展，2000，27(6)：658—661
31．祁可宗，王林安．自由基和生物抗氧化系统理论与外科学的关系．安徽农业大学学报，1996，23(2)：171—174
32. Manach C, Morand C, Texier O, et al. Quercetin metabolites in plas ma of rats fed diets containing rutin or quercetin [J].J Nutr, 1995,125:1911—1912
33. Holl man P C H, de Vires J H M, Van Lee Wen S D, et al . Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy Volunteers [J]. Am J Clin Nutr, 1995; 62:1276—1282
34. Sousc RL, et al. Arch. Biochem. Biophys, 1985: 240—345
35. T. Matsuno. Et al. Biochem. P HARMACOL, 1990,39(7):1255
36. Buckley, D.J.,et al. Influence of dietary vitamin E on the oxidative stability and quality of pigmeat. J. Anim. Sci, 1995, 73:122—130
37. Akamittath, J.G., et al. Lipid peroxidation and color stability in restructured meat systems during frozen storage. J. Food Sci, 1990, 55:1513—1517
38. T. Matsuno, et al. Biochem. Pharmacol. 1987, 36(10): 1613
39. S Beyeler, et al. Biochem, Pharmacol. 1988,37(10):1971
40. R .A .Larson .Phtochemistry ,1988 ,27 (4) :969
41. Decker, E A., et al. Inhibition of Lipid oxidation bycarnosine JAOCS, 1990,67: 650— 652
42. Maridonononneau-PariniI, et al . Pharm. Res Cpmmun, 1986, 18:61
43. MJ. Luughton, etal. Biochem. Pharmavol , 1989, 38 (17): 2859
44. Pryor, W, A, Kaufman, M, J, church, D. F, J. Drg. chem. 1985, 50 (2): 281-283
45. Halliwell B etal. chem. Toxicol, 1995, 33 (7): 601-617
46. Halliwell B and chirico S. Am J clin. Nutr, 1993, 57 (Suppl): 715-725
47. Larrauri J A, Sanchez Moreno C, Saura-Calixto F. Effect of temperature on the free
radical scavenging capacity of extracts from red and white grape pomace peels. J Agric Foo Chem, 1998, 46(7):2694-2697
48. Yokozawa T, Dong E, Natagawa T, et al. In vivo studies on the radical-scavenging activity of tea. J Agric Food Chem, 1998,46(6):2143-2150
49. Decker, E.A&Faraji, H. Imhibition of lipid by carnosine. JAOCS, 1990,67:650
50. Boldyrev, A.A.,Dupin, h.M.,Pindel, E.V.& Severin, S.E. Antioxidative properties of histidine-containing dipeptides from skeletal muscles of vertebrates. Comparative Biochemical. Physioligy, 1988,89B:245-250
51. Decker, E.A. & Crum, A.D. Inhibition of oxidative rancidity in salted ground pork by carnosine. J. Food Sci., 1991, 56:1179
52. Decker, E.A. & Crum, A.D. Antioxidant activity of carnosine in cooked ground pork, Mea Sci., 1993, 34:245
53. K M Chan, et al. Journal of Food Science , 1993, 58(1), 1—4
54. R.E. Hayes, G.N. Book Walter and E.B. Bagley. J. Food Sci.,1997,42(6)
55. Kisoon Rhee et al.J. Food Sci., 1979,44:1132
56. Hua—Ming then et al.,Structural analysis of antioxidative peptides from soybean β —conglyon. J. Agri. Food Chem.,1995,43:574—578
57. Hackler L. kastin A J. Zadina J E. Isolation of novel peptides with a unique binding profile from human brain cortex: Tyr_k_MIF_1. peptides. 1994, 15 (6): 945—950
58. Cheng D L Shao Y zhao K et al. Penpeptides from the roots of Aster tataricus. Pharmazie, 1996, 51 (1): 185—186
59. Yenhua He, Fereidoon Shahidi. Antioxidant activity of green tea and its catechins in a fish meat model system. J Agric Food Chem, 1997,45:4262-4265
60. Barry Halliwell et al. The Deroxyribose Method: A Simple of "Test=Tube"Assay For Determination of Rate Constants for Reactions Hydroxyl Radicals. Analytical Biochemistry. 1987,165:215=219
61. Kohen, R., et al. Antioxidant activity of carnosine, homocarno-sine, and anserine present in muscle and brain. Proc. Nat. Acad. Sci. USA, 1988,85:3175-3179