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1 生物工程学报 Chin J Biotech 2009, April 25; 25(4): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 食品生物技术代卉 2, 乐国伟 1,2, 孙进 2, 韩芳 2, 施用晖 1,2 1, , : 本研究旨在分析小麦蛋白活性肽对免疫抑制小鼠免疫功能和抗氧化功能的调节作用小鼠灌胃小麦肽 10 d, 第 8 天用环磷酰胺诱导免疫抑制, 测定血清溶血素抗体生成细胞含量脾细胞增殖体外腹腔巨噬细胞吞噬能力肝脏抗氧化酶活性和丙二醛 (MDA) 含量以及血清清除 DPPH 和 OH 的能力实验结果表明, 环磷酰胺处理显著的降低了小鼠血清中抗 SRBC 抗体 ( 溶血素 HC 50 ) 水平和腹腔巨噬细胞的吞噬能力 ; 同时伴随着肝脏超氧化物歧化酶活性 (SOD) 过氧化氢酶活力 (CAT) 总抗氧化能力(T-AOC) 的降低和 MDA 含量的提高给小鼠灌胃小麦肽可以恢复 HC 50 和脾细胞增殖, 显著提高抗体生成细胞含量和腹腔巨噬细胞吞噬能力 ; 此外, 小麦肽增强了小鼠血清清除 DPPH 和清除 OH 的能力以上结果表明, 小麦肽可以调节应激状态引起的机体抗氧化体系紊乱及免疫功能的降低这可能与小麦肽缓冲自由基生成激活腹腔巨噬细胞和脾淋巴细胞活性有关 : 小麦肽, 免疫抑制, 自由基, 免疫作用, 抗氧化作用 Immune modulation and antioxidant effects of wheat peptides on immunosuppressed mice Hui Dai 2, Guowei Le 1,2, Jin Sun 2, Fang Han 2, and Yonghui Shi 1,2 1 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi , China 2 School of Food Science and Technology, Jiangnan University, Wuxi , China Abstract: We studied immune modulation and antioxidant effects of wheat peptides on immunosuppressed mice. Mice were administrated with wheat peptides orally for 10 days and treated with cyclophosphamide at the 8th day. The indexes including serum hemolysin, plaque forming cells, spleen cells proliferation, liver antioxidant enzymes activties, malondialdehyde (MDA), scavenging serum 2,2-Diphenyl-1-picrylhydrazyl and OH and macrophage phagocytic ability in vitro were measured to assess the immune functions and antioxidation abilities. In vivo study shows that cyclophosphamide significantly decreases serum hemolysin (HC 50 ) and phagocytic function of macrophages. Simultaneously, liver superoxide dismutase, catalase activity and total oxidation capacity were decreased and malondialdehyde was increased. Wheat peptides could recover HC 50 and spleen cell proliferation when orally administrated. Furthermore, they could also enhance serum 2,2-Diphenyl-1-picrylhydrazyl and OH scavenging. In conclusion, wheat peptides can help body resist the stress related disorders in immune and antioxidant system. Received: November 27, 2008; Accepted: February 4, 2009 Supported by: National High Technology Research and Development Program of China (863 Program)(No. 2007AA10Z 325). Corresponding author: Yonghui Shi. Tel/Fax: ; lgw@jiangnan.edu.cn 国家高技术研究发展计划 (863 计划 )(No. 2007AA10Z 325) 资助

2 550 ISSN CN /Q Chin J Biotech April 25, 2009 Vol.25 No.4 Keywords: wheat peptides, immunosuppression, oxygen free radical, immunomodulation, antioxidation (Biologically active peptides, BAP) 6000 D, ACE,,,, [1],,,, Alcalase, 732 Sephadex G-15,,,, 1 材料与方法 1.1 试剂 Alcalase FG 2.4L (Novo.nordisk AIS 2800 Bagsvaerd): Novo ; 732 : ; Sephadex G-15: Sigma ; (SRBC): ; : ; (MTT): Sigma ; HEPES : ; R/MINI1640 : Gibco ; : ; (Cy): ; FITC : ; T-AOC MDA CAT SOD : 1.2 主要仪器 / : Zenth 3100, Anthos; MK3 : ; : ; : Thermo Forma; : ; : 1.3 实验动物 30, 18~22 g,,, : 小麦肽的制备 Alcalase, : U/g, ph 9.5, 60 o C, 4%, 100 min, 18.63%;, 732 Sephadex G-15, ( <6000 D), 1.5 动物实验 32, 4 : + (2 mg/ml) + (10 mg/ml) 20 mg/kg, 100 mg/kg, 10 ml/kg 1, 10 d, ml 2%, 2 80 mg/kg (20 mg/ml),, (PFC) [2],, /ml, 0.2% SRBC 10% 0.5 ml, 37 o C 1 h( 2, ), 3000 r/min 5 min,, 413 nm, SRBC ( HC 50 ) 100, 0 o C 1 ml 0.5 ml 10% 1 ml, 37 o C 30 min, 2000 r/min 10 min, 1 ml, 3 ml, 10 min, OD 540 OD : 0.25 ml, 4 ml, 10 min, 2000 r/min 10 min, OD 540

3 : 551 HC 50 SRBC ( ) : HC 50 =( OD /SRBC OD ) 1.5.3, /ml, 96, 0.2 ml, 4, 37 o C 5% CO 2 60 h, 1000 r/min 5 min,, MTT(5 mg/ml)20 μl, 4 h, 100 μl DMSO, OD [3], /ml, 96, 100 μl /, 37 o C 5% CO 2 4 h, 1500 r/min 10 min, 4 h, μg/, 100 μg/,, ( 6 ) 24 h, [3] FITC DPPH [4] [5] 0.1 ml, mol/l DPPH 2.8 ml, 20 min, 4000 r/min 10 min, 517 nm (A i ), (A j ) DPPH, (A 0 ),, : I(%)=(1 (A i A j )/A 0 ) 100% ( OH) [5] SOD T-AOC CAT MDA, SOD T-AOC CAT MDA 1.6 数据处理 ± ( x ± s) SPSS 10 2 结果 2.1 小麦肽对免疫抑制小鼠免疫功能的影响 (HC 50 ), (PFC) B 1 2, HC 50 (P<0.05) (P<0.01), PFC (P<0.05), (P<0.01),, HC 小麦肽对免疫抑制小鼠抗氧化能力的影响 3, DPPH (P<0.05), OH (P<0.01),, DPPH OH, (P<0.01) 4, (SOD CAT) (P<0.01), T-AOC (P<0.01), MDA (P<0.05), SOD CAT, MDA T-AOC 表 1 小麦肽对免疫抑制小鼠 HC 50 PFC 脾细胞增殖的影响 ( x ± s) Table 1 Effects of wheat peptides on HC 50, PFC, spleen lymphocytes multiplication of immunosuppressed mice ( x ± s) Group Dose (mg/kg) HC 50 PFC (OD 413 ) Spleen lymphocytes multiplication (OD 630 ) Control 54.48± ± ±0.024 Cy 20.21±0.98 * 0.22± ±0.036 Wheat peptides-l+cy ± ±0.025 * 0.19±0.015 Wheat peptides-h+cy ± ±0.025 * 0.20±0.013 Note: compare with control, * P<0.05; ** P<0.01; cy: mice were treated with cyclophosphamide (80 mg/kg); Wheat peptides-l+cy: cyclophosphamide (80 mg/kg) treated mice were administrated with low dose of wheat peptides (20 mg/kg); Wheat peptides-h+cy: cyclophosphamide (80 mg/kg) treated mice were administrated with high dose of wheat peptides (100 mg/kg).

4 552 ISSN CN /Q Chin J Biotech April 25, 2009 Vol.25 No.4 表 2 体外检测小麦肽对免疫抑制小鼠腹腔巨噬细胞吞噬功能的影响 ( x ± s) Table 2 Effects of wheat peptides on macrophage phagocytosis of immunosuppressed mice in vitro( x ± s) Group Dose (μg/well) Macrophage phagocytosis ( 10 6 ) (fluorescence intensity) Control 4.09 ± 0.42 Cy 2.05 ± 0.13 ** Wheat peptides-l+cy ± 0.22 * Wheat peptides-h+cy ± 0.58 ** Note: compare with control, * P<0.05; ** P<0.01; cy: cyclophosphamide (100μg/well); wheat peptides-l+cy: cyclophosphamide (100 μg/well)+ wheat peptides (200 μg/well); wheat peptides-h+cy: cyclophosphamide (100 μg/well)+wheat peptides (400 μg/well). 表 3 小麦肽对免疫抑制小鼠血清清除 DPPH 和清除羟自由基 ( OH) 的影响 ( x ± s) Table 3 Effects of wheat peptides on serum DPPH and OH scavenging ability of immunosuppressed mice ( x ± s) Group Dose (mg/kg) DPPH clearance rate OH clearance rate Control 27.25± ±1.80 Cy 24.42±2.23 * 29.92±4.51 ** Wheat peptides-l+cy ±1.67 ** 29.57±3.76 ** Wheat peptides-h+cy ±3.03 ** 39.89±1.56 ** Note: as the same as table 1. 表 4 小麦肽对免疫抑制小鼠体内抗氧化指标的作用 ( x ± s) Table 4 Effects of wheat peptides on antioxidant ability of immunosuppressed mice in vivo( x ± s) Group Dose (mg/kg) Liver T-AOC Liver SOD Liver MDA (nmol/mg prot) Liver CAT Control 4.15± ± ± ±1.07 Cy 2.24±0.57 ** 52.27±14.11 ** 16.52±3.56 * 20.05±2.67 ** Wheat peptides-l+cy ±0.73 * 68.89± ± ±1.44 Wheat peptides-h+cy ±0.85 ** 71.31± ± ±1.54 Note: as the same as table 1. 3 讨论, (O 2 ) OH, [6],,,,, [7],,,,,,,,,,,,,,,,,

5 : 553,, [8] Dympna [9],, REFERENCES [1] Rutherfurd-markwick KJ, Moughan PJ. Bioactive peptides derived from food. J AOAC Int, 2005, 88(3): [2] Zhang YZ, Yu LJ, Wan JM, et al. Effect of ethanol extract of lepidium meyenii walp. (Maca) on immunological function of mice. Nat Prod Res Dev, 2007, 19(2): ,,,.., 2007, 19(2): [3] Bonar A, Chmiela M, Rudnicka W, et al. Mannose-binding lectin enhances the attachment and phagocytosis of mycobacteria in vitro. Arch Immunol Ther Exp, 2005, 53(5): [4] Zhong YS, Li XC, Xie XM, et al. Study on DPPH scavenging ability of Piper longum L. J Liaoning Univ TCM, 2007, 9(1): ,,,. DPPH., 2007, 9(1): [5] Huang HL, Xu B. Free radical scavenging activities and principals of Gueldenstaedtia multiflora Bge. J Qingdao Univ (Nat Sci Ed), 2006, 19(4): ,.. ( ), 2006, 19(4): [6] Maccarrone M, Ullrich V. Redox regulation in disease and ageing. Cell Death Differ, 2004, 11(8): [7] Djordjević VB. Free radicals in cell biology. Int Rev Cytol, 2004, 237: [8] Valavanidis A, Vlahogianni T, Dassenakis M, et al. Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants. Ecotoxicol Environ Saf, 2006, 64 (2): [9] Morrow DM, Entezari-Zaher T, Romashko J, et al. Antioxidants preserve macrophage phagocytosis of Pseudomonas aeruginosa during hyperoxia. Free Radical Biol Med, 2007, 42(9): 生物工程学报对摘要的写作要求 1. (Purpose) (Methods) (Results) (Conclusions) 2., 3. (1) (2) (3) (4) (5) DNA ATP (6) (7)

中草药 Chinese Traditional and Herbal Drugs 第 46 卷第 9 期 2015 年 5 月 1339 非酒精性脂肪性肝病 (nonalcoholic fatty liver disease,nafld) 是一种与胰岛素抵抗遗

中草药 Chinese Traditional and Herbal Drugs 第 46 卷第 9 期 2015 年 5 月 1339 非酒精性脂肪性肝病 (nonalcoholic fatty liver disease,nafld) 是一种与胰岛素抵抗遗 1338 中草药 Chinese Traditional and Herbal Drugs 第 46 卷第 9 期 2015 年 5 月九味肝泰胶囊对高脂饮食诱导大鼠非酒精性脂肪肝的治疗作用陈菲 1 2,, 艾国 3, 盛柳青 1, 麻佳蕾 1, 黄正明 1. 金华职业技术学院医学院, 浙江金华