摘要
蓝莓花青素是蓝莓中天然存在的一类生物活性物质,具有重要的生理功能和开发应用前景。为实现蓝莓花青素的综合利用,本文以栽培的蓝莓果(品种为爱国者)研究对象,在蓝莓花青素分离纯化的基础上,对制备的花青素单体进行了结构鉴定。并通过建立急性小鼠肝损伤模型,探讨了蓝莓花青素对四氯化碳(CCl4)诱导小鼠的肝损伤的保护和氧化应激的影响,结合其体外清除自由基抗氧化活性和对CCl4诱导的L-02肝细胞损伤的保护,从抗氧化的角度探讨了蓝莓花青素对肝损伤保护作用的主要机制。主要研究结果如下：
(1)蓝莓果中花青素类物质的最佳提取工艺参数为：0.5%三氟乙酸-甲醇为提取溶剂,料液比为1：15,p H为2.5,加酶量3%,酶解温度70℃,酶解反应时间1h。在此条件下,经HPLC法测定,鲜果中花青素含量为5.65mg/g。
Amberlite XAD-7大孔树脂对蓝莓花青素表现出良好的吸附与解吸性能,是分离纯化蓝莓花青素的理想树脂。Amberlite XAD-7树脂的优化动态吸附条件为：蓝莓花青素液浓度2.5mg/mL, pH2.5,进样速率1.0mL/min;优化洗脱条件为：甲醇浓度70%,洗脱速率1.0mL/min,洗脱体积10BV。在此条件下,Amberlite XAD-7树脂对蓝莓花青素的动态吸附量达最大值18.5mg/g,动态解吸回收率达87.15%。
通过制备型高效液相色谱制备出三种主要的花青素单体,经过结构鉴定,组分1为锦葵色素-3-半乳糖苷(M3G),纯度为95.49%：组分3为矢车菊素-3-葡萄糖苷(C3G),纯度为98.57%；组分6为矢车菊素-3-芸香苷(C3R),纯度为96.34%。这三种单体占花青素的含量分别为12.76%、60.68%和15.87%。
(2)蓝莓花青素具有一定的抗氧化活性,在DPPH自由基清除实验和β-胡萝卜素／亚油酸自氧化体系中,相同浓度下,其作用效果优于抗坏血酸。蓝莓花青素分离的三种单体总抗氧能力表现为C3G>C3R>M3G,且半抑制浓度(IC50值)表明,矢车菊素-3-葡萄糖苷(C3G)清除过氧烷基及DPPH自由基的能力最强(P<0.05),从而进一步说明其为蓝莓花青素中有效的抗氧化活性成分之一。
(3)小鼠摄入蓝莓花青素后的低(0.5g/kgbw)、中(1.0g/kgbw)、高(2.0g/kg bw)剂量组经CCl4诱导的急性肝损伤小鼠的肝酶活性较模型组显著降低(P<0.05),血清和肝脏中丙二醛(MDA)的生成量显著减少(P<0.05),超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性明显增强(P<0.05),肝脏组织的总抗氧化能力(T-AOC)显著提高(P<0.05)：由CCl4引起的肝脏组织气球样变、脂肪变性、炎症浸润等病理学损伤,喂食蓝莓花青素后,均可得到明显改善。
(4)通过建立CCl4对人胚胎肝细胞株L-02的损伤模型,结果表明：CCl4诱导L-02肝细胞损伤的最适损伤浓度为20mmol/L,(?)员伤时间为6h。经CCl4损伤后模型组细胞存活率仅为45.68%,上清液肝酶活性显著升高(P<0.05),脂质过氧化产物MDA含量显著增加(P<0.05)。蓝莓花青素可一定程度地改善L-02肝细胞损伤,能明显提高肝细胞的存活率,减少肝酶的释放,存在明显剂量效应关系(P<0.05)。蓝莓花青素中的三种主要成分单体的抗肝损伤体外细胞试验结果表明,C3G对提高细胞存活率和细胞抗氧化水平作用最好,且与其它两种单体间存在显著性差异(P<0.05)。最终证明了蓝莓花青素中有效的抗肝损伤活性单体为矢车菊素-3-葡萄糖苷(C3G)。
细胞克隆形成抑制实验、细胞周期DNA含量分析法及Annexin V-FITC/PI双染色检测法的结果表明,矢车菊素-3-葡萄糖苷保护L-02正常肝细胞的形式是减少细胞的坏死。免疫印迹法检测调控细胞凋亡中的关键蛋白Caspase-3的含量变化进一步说明,矢车菊素-3-葡萄糖苷抑制L-02细胞因损伤而坏死的机理为Caspase依赖型,而细胞损伤可能是通过线粒体相关途径实现的坏死过程。
Blueberry anthocyanins are one kind of important bioactivators in blueberries, having many kinds of significant physiological functions and broad prospects of development and application. In order to gain insight into the comprehensive utilization of blueberry anthocyanins, we studied blueberries in the northeast and identified its anthocyanins monomers structures on the basis of isolation and purification of blueberry anthocyanins. With acute hepatic injury of mice by CCl4model established, the potential protection of blueberry anthocyanins was demonstrated. Otherwise, with the research of antioxidant activities of blueberry anthocyanins in vitro and protective effect of blueberry anthocyanins on L-02injured by CCl4. The protective effect and antioxidant mechanism of blueberry anthocyanins on hepatic injury of mice induced by CCl4was further discussed; Results were as follows:
(1) The optimum enzyme assisted extracting condition of anthocyanin compounds from blueberry:0.5%trifluoroacetic acid-methanol as the extraction solvent, solid-liquid ratio of1:15, p H2.5, added3%of the amount of enzyme, hydrolysis temperature70℃, the enzymatic reaction time1h. Under these conditions, the yield of blueberry anthocyanins was5.65mg/g, measured by RP-HPLC method.
Amberlite XAD-7macroporous adsorptive resin had good adsorption and desorption capability. The optimum dynamic adsorption condition of Amberlite XAD-7was as follows:blueberry anthocyanins concentration2.5mg/mL, pH value2.5, velocity of flow1.0mL/min. The optimum desorption condition was as follows:methanol concentration70%, velocity of flow1.0mL/min, desorption volume10BV. Under these parameters, the maximum dynamic adsorption quantity of Amberlite XAD-7resin for blueberry anthocyanin was up to18.5mg/g, dynamic desorption rate was87.15%.
Three main anthocyanins monomers were got by the preparation of HPLC, through the structure identification, No.1component was Malvidin-3-galactoside (M3G), with the purity of95.49%; No.3component was Cyanidin-3-glucoside(C3G), with the purity of98.57%; No.6component was Cyanidin-3-rutinoside(C3R), with the purity of96.34%. The three monomers were respectively made up12.76%,60.68%and15.87%of total anthocyanin contents.
(2)Blueberry anthocyanins have certain antioxidant activity, whose effect was better than than vitamin C in the same concentration in DPPH radical scavengingassay and β-carotin/linolic acid oxidation inhibiting activity. The order of antioxidant activity of three blueberry anthocyanins monomers was C3G>C3R>M3G.While the values result of IC50was showed that cyanidin-3-glucoside(C3G) exhibited the strongest response in alkyl peroxide and DPPH radical scavenging. All the results turned out that cyanidin-3-glucoside was one of the most active antioxidant ingredients in blueberry anthocyanin.
(3) When the mice was treated with blueberry anthocyanins(in dosages of0.5g/kg bw,1.0g/kg bw,2.0g/kg bw), the activity of ALT was significantly decreased compared with model group (P<0.05), AST was also decreased(P<0.05); MDA, the product of lipid peroxidation was decreased(P<0.05); Meanwhile, the activities of antioxidant enzymes, such as SOD and GSH-Px were enhanced (P<0.05); The pathology damage of the liver indueed by CCl4, such as balloon-like change, fatty degeneration.inflammatory infiltration were all improved obviously.
(4) To establish liver cell injury model, the concentration of CCl4was20mmol/L, and the time was6h. In the model group, the L-02cell survival rate were decreased to45.68%, while the liver enzyme activities were enhanced and the content of MDA was increased significantly(P<0.05). The injury of L-02was retarded by blueberry anthocyanins. Compared to cell in model group, the cell survival rate was increased, and the liver enzyme was reduced, the more the concentration, the higher the survival rate(P<0.05). Results showed the obvious dose-response relationship. Furthermore, liver damage protection effect of the three main ingredients of blueberry anthocyanins (C3G, C3R and M3G) on L-02cells were investgated. The results showed that C3G had the best effect to improve the level of cell survival and cell anti-oxidation, and there were significant difference between the others(P<0.05), Cyanidin-3-glucoside(C3G) was the most effective anti-damage active monomer.
Result of cell colony formationinhibitory assay, cell cycle progress and Annexin V-FITC/PI staining analysis showed that Cyanidin-3-glucoside protected L-02cells by reducing cell necrosis. Contents change of cell apoptosis regulation protein Caspase-3detected by western blotting demonstrated that the cell necrosis caused by injury was Caspase-dependent in L-02cells, while necrosis of cells might be via mitochondria pathway.
引文
[1]方忠祥,倪元颖,陈欣.花青素生理功能研究进展[J].食品工业科技,2001,17(3)：60-62.
[2]王兆雨,徐美玲,朱蓓薇,等.蓝莓花青素的提取工艺条件IJ].大连轻工业学院学报,2007,9(3)：123-128.
[3]王庆,韩平华,张名位,等.膳食补充黑米皮对冠心病患者血浆中氧化应激、炎症和脂质水平的影响[J].中山大学学报(医学科学版),2007,28(3)：301-305.
[4]王威,陈景红,王新宁,等.葡萄籽原花青素诱导人肝癌细胞凋亡及自噬性死亡[J].暨南大学学报(医学版),2011,32(2)：181-185.
[5]卢林耿,王中民,张永祥,等.对四氯化碳所致大鼠肝脏损伤机制的研究[J].中西医结合肝病杂志,2009,6(2)：24-30.
[6]宁正祥,李卫.花青素类化合物的分子修饰[J].食品科学,2011,26(8)：505-507.
[7]向平,林益明,林鹏,等.基质辅助激光解吸附飞行时间质谱分析缩合单宁的阳离子化问题[J].分析化学,2006,34(7)：1019-1022.
[8]向道丽.酶法提取越橘果渣花色苷酶解条件的研究[Jj.中国林副特产,2005,12(6)：123-125.
[9]孙芸,谷文英.硫酸-香草醛法测定葡萄籽原花青素含量[J].食品与发酵工业,2003,29(9)：43-46.
[10]孙怡,商学军.花青素诱导前列腺癌PC-3细胞凋亡的机制研究[J].安徽中医学院学报,2009,28(1)：47-49.
[11]朱凤妹,杜彬,李军,等.大孔吸附树脂分离葡萄酒下脚料中原花青素的研究[J].食品工业,2008,15(1)：6-9.
[12]严琼琼,唐书泽,孙承锋.苹果多酚对冷却猪肉腐败菌抑菌效果的影响[J].食品研究与开发,2009,30(10)：117-121.
[13]张苏云.凝胶层析分离提纯芦荟多糖的方法[J].食品工业科技,2008,11：208-212.
[14]张泽生,徐英,李欣.大孔吸附树脂对苹果渣中苹果多酚吸附性能的研究[J].食品研究与开发,2006,27(9)：24-27.
[15]张玲,郭晓雷,张秋影.紫杉醇-抗HER2MAb偶联物体外抗肿瘤活性的研究[J].中国实验诊断学,2010,14(7)：17-32.
[16]张甜,董学畅,吴方评,等.微柱高效液相色谱和质谱法测定烟草样品中多酚[J].理化检验-化学分册,2005,41(5)：7-10.
[17]张静,连超群,陈传好,等.姜黄素诱导A549细胞凋亡过程中活性氧水平和caspase-3活性的变化[J].中国老年学杂志,2011,6(31)：2024-2028.
[18]李丹,林琳.越桔食品资源的开发与利用[J].食品与发酵工业,2009,26(4)：76-81.
[19]李亚东,张志东,吴林.蓝莓果实的成分及保健机能[J].中国食物与营养,2002,(1)：27-28.
[20]李连达,靖雨珍.中国保健食品的优势与发展[J].中国工程科学,2003,5(5)：35-39.
[21]李建新,王娜,王海军,等.苹果多酚的减肥降脂作用研究[J].食品科学,2008,29(8)：597-599.
[22]李春阳,许时婴,王璋DPPH法测定葡萄籽花青素清除自由基的能力[J].食品与生物技术学报,2006,25(2)：102-106.
[23]李春阳,许时婴,王璋.低浓度盐酸-香草醛法测定葡萄籽中原花青素含量的研究[J].食品工业科技,2004,25(6)：128-130.
[24]李春阳,张红城,靖雨珍,等.激光光散射与GPC联用测定葡萄籽原花青素相对分子质量[J].江苏农业学报,2009,25(6)：1354-1359.
[25]李莹,药立波,韩炯.葡萄籽提取物花青素诱导胃癌细胞脱落调亡[J].中国药理学通报,2004,20(7)：761-764.
[26]李婷婷,宋述尧,迟燕平.高压脉冲电场对大蒜采后抗氧化物质的影响[J].食品科学,2009,30(6)：272-274.
[27]汪明明,崔速南,刘靓雯,等.外周血淋巴细胞穿孔素和颗粒酶在不同乙型肝炎患者中的表达率比较[J].中华微生物学和免疫学杂志,2007,27(9)：855-859.
[28]肖湘,卢刚,张捷,等.黑色食品色素清除活性氧功效及抗氧化活性[J].药物生物技术,2000,7(2)：112-115.
[29]肖嶙,万会师.酶在植物有效成分提取中的应用[J].安徽农业科学,2006,34(8)：1551-1552.
[30]陈健,孙爱东,高雪娟,等.蓝莓花青素的提取及抗氧化性的研究[J].北京林业大学报,2011,33(2)：126-130.
[31]陈笳鸿,汪咏梅,吴冬梅,等.没食子酸与纤维素的酯化合成及产物功能特性试验[JJ.林产化学与工：业,2005,25(2)：6-10.
[32]陈磊,王军.葡萄籽中原花青素的提取[J].中国野生植物资源,2008,27(1)：58-60.
[33]金莹,孙爱东,陈健.苹果多酚对小鼠H22细胞株移植性肿瘤的抑制作用[J].安徽农业科学,2010,38(24)：13525-13526.
[34]金莹,孙爱东,陈健.大孔树脂纯化苹果多酚的研究[J].食品科学,2007,28(4)：160-163.
[35]段玉清,张海晖,唐瑛,等.莲房花青素对黑色素瘤B16体内抑制作用的研究[J].中成药,2007,29(12)：1828-1830.
[36]唐传核,彭志英.天然花色苷类色素的生理功能及应用前景[J].冷冻与速冻食品工业,2000,6(1)：26-28.
[37]夏效东,凌文华,夏敏,等.黑米花色苷提取物对ApoE基因缺陷小鼠动脉粥样硬化晚期斑块的影响[JJ.食品科学,2006,27(3)：213-215.
[38]徐曼,陈笳鸿,汪咏梅,等.落叶松花青素的没食子酰化及其抗氧化活性增强效应[J].林产化学与工业,2010,30(6)：55-60.
[39]贾艳菊,代玲,张灿.龙葵生物碱诱导HeLa细胞凋亡的研究.动物医学进展[J].食品科学,2010,31(8)：51-54.
[40]郭红辉,胡艳,刘驰,等.黑米花色苷对果糖喂养大鼠的抗氧化及胰岛素增敏作用[J].营养学报,2008,30(1)：85-87.
[41]陶令霞,王浩,常慧萍,等.苹果皮渣中苹果多酚的超声辅助提取工艺优化及其抗脂质氧化活性研究[J].河南工业大学学报(自然科学版),2008,29(1)：32-36.
[42]高永贵,王岳飞,杨贤强,等.脂溶性茶多酚抗油脂氧化及其增效剂的研究[J].2010,5(2)：30-32.
[43]常徽,糜漫天,凌文华.黑米花色素及联合化疗药物对不同肿瘤细胞增殖的影响[J].第三军医大学学报,2007,29(20)：1943-1946.
[44]焦中高,刘杰超,王思新.黑莓红色素抗氧化活性研究[J].食品科技,2003,(8)：63-65.
[45]蒋莉,李跃华,戚晓红,等.丹参素对内毒素性肝损伤的防护作用及其机制的研究[J].中西医结合肝病杂志,2009,9(2)：30-32.
[46]谭天伟.天然产物分离新技术[J].化工进展,2003,22(7)：665-668.
[47]颜流水,井晶,黄智敏,等.槲皮素分子印记聚合物的制备及固相萃取性能研究[J].分析试验室,2006,25(5)：97-100.
[48]魏福祥,韩菊,王改珍,等.低聚原花青素提取技术研究[J].食品工业科技,2003,14(9)：54-55.
[49]魏福祥,韩菊,张兰,等.葡萄籽中提取低聚原花色素的技术研究[J].现代化工,2001,21(4)：27-29.
[50]Almeida JR, Damico E, Preuss A, et al. Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria ×ananassa)[J]. Archives of Biochemistry and Biophysics,2007,465(1):61-71.
[51]Anna R, Renato CN, Daniela V, et al. Modification of glass transition temperature through carbohydrates addition and anthocyanin and soluble phenol stability of frozen blueberry juices [J]. Journal of Food Engineering,2003,56(3):229-231.
[52]Annanaryju D, Sarma E. Antioxidant ability of anthocyanins against ascorbic acid oxidation[J]. Phyto-chemistry,2007,45(4):671-674.
[53]Astadi IR, Astuti M, Santoso U, et al. In vitro antioxidant activity of anthocyanins of black soybean seed coat in human low density lipoprotein (LDL) [J]. Food Chemistry,2009,112(3): 659-663.
[54]Awika J, Rooney L, Waniska R. Anthocyanins from black sorghum and their antioxidant properties. Food Chemistry,2004,90(12):293-301.
[55]Ayman EH, Shesha HJ. Effect of biological cell size and shape on killing efficiency of pulsed electric field. IEEE International Conference on Dielectric Liquids.2008.
[56]Chen J, Sun HN, Sun AD, et al. Studies of the protective effect and antioxidant mechanism of blueberry anthocyanins in a CCl4-induced liver injury model in mice[J]. Food and agricultural immunology,2012,23(4):352-362.
[57]Chen ZY, Zhu QY, Tsang D, et al. Degradation of green tea catechins in tea drinks [J]. Journal of Agricultural and Food Chemistry,2011,49(1):47-482.
[58]Du Q, Zheng J, Xu Y. Composition of anthocyanins in mulberry and their antioxidant activity. Food Composition and Analysis.2008,21(5):390-395.
[59]Elisia L, Hu Ch, Popovich DG. Antioxidant assessment of an Anthocyanin-enriched blackberry extract.Food Chemistry,2007,101 (8):1052-1058.
[60]Erhardt A, Biburger M, Papadopoulos T. IL-10, regulatory T cells, and Kupffer cells mediate tolerance in concanavalin A-induced liver injury in mice. Hepatology,2007,45(2):475-485.
[61]Evrendilek GA, Zhang QH. Effects of pulse polarity and pulsed delaying time on pulsed electric fields-induced pasteurization off. coli O157:H7. Journal of Food Engineering,2005,68(4):271-276.
[61]Faria A, Calhau C, Defreitas V, et al.Anthocyanins as antioxidants and tumor cell growth modulators [J]. Journal of Agricultural and Food Chemistry,2011,54(6):2392-2397.
[62]Francisco T, Clara C, Maria JE. Effect of high-intensity pulsed electric fields processing and conventional heat treatment on orange-carrot juice carotenoids.Journal of Agricultural and Food Chemistry,2005,53(12):9519-9525.
[63]Gonzalez FJ. Role of cytochromes P450 in chemical toxicity and oxidative stress:studies with CYP2E1[J]. Mutation Research,2005,569(1):101-110.
[64]Gorettaa LA, Romancxykb LJ, Rodrigueza CB, et al. Cytotoxic effects of digalloyl dimer anthocyanins in human cancer cell lines [J]. Nutritional Biochemistry,2008,19(12):797-808.
[65]Gungor N, Sengul M. Antioxidant activity, total phenolic content and selected physicochemical properties of white mulberry (Morus alba L.) fruits.International Journal of Food Properties, 2008,11(8):44-52.
[66]Ha BJ, Lee JY. The effect of chondroitin sulfate against CCl4-induced hepatotoxicity [J]. Biological and Pharmaceutical Bulletin,2010,26(5):622-626.
[67]He XJ, Liu RH. Phytochemicals of Apple Peels:Isolation, Structure Elucidation, and Their Antiproliferative and Antioxidant Activities[J]. Journal of Agricultural and Food Chemistry,2008, 56(6):9905-9910.
[68]Hyun JR, In YB, Torchio F, et al. Enhancement of anti-radical activity of pectin from apple pomace by hydroxamation[J]. Food Hydrocolloids,2011,25 (3):545-548.
[69]Hsiao G, Shcn MY, Lin KH, et al. Inhibitory activity of kinetin on free radical formation of activated platelets in vitro and on thrombus formation in vivo. European Journal of Pharmacology, 2003,465(3):281-287.
[70]Hsu CL, Huang SL, Yen GC, et al. Inhibitory effect of phenolic acids on the proliferation of 3T3-L1 preadipocytes in relation to their antioxidant activity[J]. Journal of Agricultural and Food Chemistry,2006,54(9):4191-4197.
[71]Hu C, Zawistowski J, Ling W, et al. Black rice (Oryzasativa L. indica) pigmented fraction suppresses both reactive oxygen species and nitric oxide in chemical and biological model systems. Journal of Agricultural and Food Chemistry,2003,51(4):5271-5277.
[72]Hu JJ, Yoo JS, Lin M, et al. Protective effects of diallyl sulfide on acetaminophen-induced toxicities. Food and Chemical Toxicology,1996,34(6):963-969.
[73]Hu XY, Meng G, BaoY Y, et al. Relationships between induction of apoptosisby Taxol in Hela cells and apoptosis-related proteins[J]. Chinese Pharmacological Bulletin,2010,20 (9):1063-1072.
[74]Iked AR, Che XF, Yamaguchi T, et al. Cepharant hine potently enhances the sensitivity of anticancer agent s in K562cells[J]. Cancer Science,2009,86 (6):372-376.
[75]Jayathilakan K, Sultana K, Radhakrishna K, et al. Effect of irradiation on differential scanning calorimetric profile of fluidised bed dried mutton. International Journal of Food Properties,2012,15(5):202-210.
[76]Jing H, Kitts DD. Antioxidant activity of sugar-lysine Maillard reaction products in cell free and cell culture systems[J]. Archives of Biochemistry and Biophysics,2004,429(7):154-163.
[77]Jin ZL, ZhangHQ, Li S, et al. Quality of apple sauces processed by pulsed electric fields and HTST pasteurization. International Journal of Food Science and Technology,2009, 44(8):829-839.
[78]Kang Y, Lee DC, Han J. NM23-H2 involves in negative regulation of Diva and BCl-2L10 in apoptosis signaling[J].Biochemical Biophysical Research Communications,2010,359(1):76-82.
[79]Kelly LW, Kang XMi, Joseph GS, et al. Cellular Antioxidant Activity of Common Fruits[J]. Journal of Agricultural and Food Chemistry,2008,56(5):8418-8426.
[80]Kim JH, Yoon S, Won M. HIP1R interaets with a member of Bcl-2 family, BCl-2L10, and induees BAK-dependent cell death[J]. Cellular Physiology and Biochemistry,2009,23(3):43-52.
[81]Lam WH, Kazi A, Kuhn DJ. A potential prodrug for a green tea polyphenol proteasome inhibitor: evaluation of the peracetate ester of (-)-epigallocatechin gallate [(-)-EGCG] [J]. Bioorganic &Medicinal Chemistry,2010,12(21):5587-5593.
[82]Legua P, Melgarejo P, Martinez JJ, et al. Evaluation of spanish pomegranate juices:organic acids, sugars, and anthocyanins. International Journal of Food Properties,2012,15(3):481-494.
[83]Liu M, Li XQ, Weber C, et al. Antioxidant and antiproliferative activities of raspberries [J]. Journal of Agricultural and Food Chemistry,2002,50(2):2926-2930.
[84]Lykkesfeldt J. Malondialdehyde as biomarker of oxidative damage to lipids caused by smoking. Clinica Chimica Acta,2007,380(1):50-58.
[85]Maldonado-Celis ME, Souad B, Francine G. Apple anthocyanins activate apoptotic signaling pathway in human colon adenocarcinoma cells by a lipid raft independent mechanism [J]. Biochemical and Biophysical Research Communications,2009,388(2):372-376.
[86]Matsumoto H, Nakamura Y, Tachibanaki S, et al. Stimulatory effect of cyanidin 3-glycosides on the regeneration of rhodopsin. Journal of Agricultural and Food Chemistry,2003, 51(5):3560-3563.
[87]Mazza G, Kay CD, Cottrell T, et al. Absorption of anthocyanins from blueberries and serum antioxidant status in human subjects. Journal of Agricultural and Food Chemistry, 2002,50(3):7731-7737.
[88]Molan AL, Lila MA, Mawso NJ. Satiety in rats following blueberry extract consumption induced by appetite-suppressing mechanisms unrelated to in vitro or in vivo antioxidant capacity[J]. Food Chemistry,2008,107(3):1039-1044.
[89]Moore J, Yin J, Yu L, et al. Identification and characterization of methylated and ring-fission metabolites of tea catechins formed in humans, mice, and rats [J]. Journal of Agricultural and Food Chemistry,2006,54(5):617-626.
[90]Osanai K, Huo C, Landis-Piwowar KR, et al. Synthesis of(2R,3R)-epigallocatechin-3-O-(4-hydroxybenzoate), a novelcatechin from Cistus salvifolius, and evaluation of itsproteasome inhibitory activities [J]. Tetraedron,2011,63(32):7565-7570.
[91]Palmes D, Spiegel HU. Animal models of liver regeneration. Biomaterials,2004,25(9):1601-1611.
[92]Paola M, Alexandre S, Maria JE, et al. Determination of phenolic compounds in Yucca gloriosa bark and root by LC-MS [J]. Journal of Pharmaceutical and Biomedical Analysis,2008,47(7): 854-859.
[93]Pavagadhi S, Joseph GS, Jena BS. Antioxidant principles in peltophorum ferrugineum flower extracts.International Journal of Food Properties,2012,15(3):549-557.
[94]Renaud S, Lorgeril MD. Wine, alcohol, platelets, and the French paradox for coronary heart disease. Lancet,1992,339(9):1523-1526.
[95]Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation duringapoptosis[J]. Nature Reviews Molecular Cell Biology,2011,5(11):897-907.
[96]Rong T, Raymond Y. Optimization of a new mobile phase to know the complex and real polyphenolic composition:towards a total phenolic index using high-performance liquid chromatography [J]. Journal of Chromatography A,2003,18(1):29-40.
[97]Rolle L, Torchio F, Ferrandino A, et al. Influence of wine-grape skin hardness on the kinetics of anthocyanin extraction. International Journal of Food Properties,2012,15(7): 249-261.
[98]Sabine S, Gerhard D, Katri M, et al. Dietary fiber-rich colloids from apple pomace extraction juices do not affect food intake and blood serum lipid levels, but enhance fecal excretion of steroids in rats[J]. The Journal of Nutritional Biochemistry,2004,15(5):296-302.
[99]Salda AG, PuertolasE, LopezN. Comparing the PEF resistance and occurrence of sublethal injury on different strains of Escherichia coli, Salmonella Typhimurium, Listeria monocytogenes and Staphylococcusaureus in media of pH 4 and 7. Innovative Food Science and Emerging Technologies,2009,10(1):160-165.
[100]Sang S, Lambert JD, Yang CS. Bioavailabihty and stability issues in understanding the cancer preventive effects of tea polyphenols [J]. Journal Science of Food and Agriculture,2006,86(14): 2256-2265.
[101]Sanchez-Moreno C, Ancos DB, PlazaL, et al. Nutritional approaches and health-related properties of plant foods processedby high pressure and pulsed electric fields. Critical Reviews in Food Science and Nutrition,2009,49(7):552-576.
[102]Shanna SD, Katiyar SK. Dietary grape-seed proanthocyanidin inhibition of ultraviolet B-induced immune suppression is associated with induction of IL-12[J]. Carcinogenesis,2006,27(1): 95-102.
[103]Siddiq M, IezzoniA, Khan A, et al. Characterization of new tart cherry (Prunus cerasus L): selections based on fruit quality, total anthocyanins, and antioxidant capacity. International Journal of Food Properties,2011,14(10):471-480.
[104]Siebler J, Wirtz S, Frenzel C, et al. Cutting edge:a key pathogenic role ofIL-27 in T cell-mediated hepatitis. The Journal of Immunology,2008,180:30-33.
[105]Siriwoharn T, Wrolstad RE, Durst RW. Identification of ellagic acid in blackberry juice sediment. Journal of Food Science,2005,70(13):189-197.
[106]Son YO, Kim J, Lim JC, et al. Ripe fruit of Solanum nigrumL. inhibits cell growth and induces apoptosis in MCF-7 cell [J]. Food and Chemical Toxicology,2009,41 (10):1421-1428.
[107]Sun J, Liu RH. Apple phytochemical extracts inhibit proliferation of estrogen-dependent and estrogen-independent human breast cancer cells through cell cycle modulation [J]. Journal of Agricultural and Food Chemistry,2008,56 (24):11661-11667.
[108]Tate P, Kuzmar A, Smith SW, et al. Comparative effects of eight varieties of blackberry on mutagenesis. Nutrition Research,2003,23(4):971-979.
[109]Tang HD, Li C, Wang L, et al.Granzyme H of cytotoxic lymphocytes is required for clearance of the hepatitis B virus through cleavage of the hepatitis B Virus X protein [J]. Journal of Immunology,2012,188(2):824-831.
[110]Tanida I, Minematsu-Ikeguchi N, Ueno T. Lysosomal turnover, but not a cellular level of endogenous LC3 is a marker for autophagy. Autophagy,2005,1(4):84-91.
[111]Tian Q, Giusti MM, Stoner GD, et al. Screening for anthocyanins using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry with precursor-ion analysis, product-ion analysis, common-neutral-loss analysis,and selected reaction monitoring. Journal of Chromatography. A,2005,91(10):72-82.
[112]Toshihiko SJ, Saeko M, Nina M, et al. Take Apple (Malus pumila) procyanidins fractionated according to the degree of polymerization using normal-phase chromatography and characterized by HPLC-ESI/MS and MALDI-TOF/MS. J. of Chromatography A,2006,1102(8):206-213.
[113]Tsuda T, Ueno Y, Kojo H, et al. Microarray profiling of gene expression in human adipocytes in response to anthocyanins[J]. Biochemical Pharmacology,2006,71(15):1184-1197.
[114]Walkling-Ribeiro M, Noci F, Cronin DA, et al. Reduction of staphylococcusaureus and quality changes in apple juice processed by ultraviolet irradiation, pre-heating and pulsed electric fields. Journal of Food Engineering,2008,89(2):267-273.
[115]Wang J, Mazza G. Inhibitory effects of anthocyanins and other phenolic compounds on nitric oxide production in LPS/IFNgamma-activated RAW 264.7 macrophages. Journal of Agricultural and Food Chemistry,2002,50(3):850-857.
[116]Wang L, Zhang K, WuL, et al. Structural insights into the substrate specificity of human granzyme H:the functional roles of a novel RKR motif [J]. Journal of Immunology,2012,188(2): 765-773.
[117]Wang SY, Lin HS. Antioxidant activity in fruits and leaves of blackberry, raspberry, and strawberry varies with cultivar and developmental stage. Journal of Agricultural and Food Chemistry,2000,48(6):140-146.
[118]Wolfe KL, Liu RH. Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. Journal of Agricultural and Food Chemistry,2007,55(14):8896-8907.
[119]Wu X, Cao G, Prior RL. Absorption and metabolism of anthocyanins in elderly women after consumption of elderberry or blueberry. Journal of Nutrition,2002,132(2):1865-1871.
[120]Yan M, Xu Q, Zhang P, et al. Correlation of NF-kappaB signal pathway with tumor metastasis of human head and neck squamous cell carcinoma [J], BMC Cancer,2010,10(1):437-449.
[121]Yang YP, Stephen AK, Gandhi CR. Kupffer cells are a major source of increased platelet activating factor in the CCl4-induced cirrhotic rat liver. Journal of Hepatology,2003,39(2): 200-207.
[122]Yi W, Akoh CC, Fischer J, et al. Effects of phenolic compounds in blueberries and muscadine grapes on HepG2 cell viability and apoptosis. Food Research International,2006,39(13): 628-638.
[123]Zenkevich IG, Rodin AA. One-Stage Determination of the Number of Hydroxyl Groups in Polyatomic Phenols by Gas Chromatography Using Mixed Reagents[J]. Journal of Analytical Chemistry,2002,57 (7):611-615.
[124]Zhang FJ, Yang JY, Mou YH, et al. Inhibition of U-87 human glioblastoma cell proliferation and formylpeptide receptor function by oligomer procyanidins (F2) isolated from grape seeds. Chemico Biological Interactions,2009,179(12):419-429.
[125]Zhang LI., Lin YM. HPLC, NMR and MALDI-TOFMS analysis of condensed tannins from Lithocar Pus glaber leaves with Potent free radical scavenging activity[J]. Molecules,2008, 13(12):2986-2997.
[126]Zhang Z, Kou X, Fugal K, et al. Comparison of HPLC methods for determination of anthocyanins and anthocyanidins in bilberry extracts. Journal of Agricultural and Food Chemistry, 2004,52(6):688-691.
[127]Zhong K, Wu JH, Wang ZF, et al. Inactivation kinetics and secondary structural change of PEF-treated HRP and PPO[J]. Food Chemistry,2006.104(2):483-491.