鲑鱼皮明胶水解物的抗氧化活性及其对细胞氧化损伤的保护作用

采用碱性蛋白酶、木瓜蛋白酶和中性蛋白酶水解鲑鱼皮明胶,得到水解度为4.7％到13.5％的7个明胶水解物,分别评价明胶水解物对自由基的清除能力.明胶水解物(0.5、1、2mg/mL)预先作用BRL大鼠肝细胞2h后,通过H2O2(5mmol/L)诱导氧化损伤,分别测定细胞存活率、乳酸脱氢酶(LDH)、丙二醛(MDA)和谷胱甘肽(GSH)等细胞内抗氧化产物的含量变化.结果表明,明胶水解物的抗氧化活性随着水解度的增大呈增强趋势.明胶水解物对细胞具有保护作用,可显著提高细胞存活率,降低LDH渗出量和MDA生成量,且存在一定的剂量关系,但细胞中GSH含量变化不显著;7个明胶水解物对H2O2诱导肝细胞损伤保护时,细胞存活率与水解物的体外抗氧化活性大小显著正相关,而LDH渗出量和MDA生成量与水解物的体外抗氧化活性大小负相关.     

4种水溶性维生素对酪蛋白氧化的保护作用

酪蛋白是乳与乳粉中的主要蛋白组分,在加工和贮藏过程中会发生氧化反应,从而影响产品品质,因此应有效防控以乳蛋白为基料的食品的品质劣变。本文研究了Fenton体系诱导的酪蛋白氧化及添加维生素C(VC),VB_6,VB_2,VB_(12)对氧化酪蛋白的保护作用。采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳检测氧化酪蛋白各组分条带的变化,测定氧化酪蛋白的结构及功能特性的变化,包括巯基含量、羰基含量及溶解性、蛋白变性程度。结果表明,随Fenton体系中Fe2+及VC的浓度增大,氧化后酪蛋白4个组分条带的密度均逐渐减小,并在相应的高分子区域出现逐渐致密的新条带;氧化使酪蛋白的结构与功能特性均发生变化:溶解度和总巯基含量显著降低;变性程度和羰基含量显著升高。VC≥6 mmol/L,VB_61~12 mmol/L,VB_20.1~0.8 mmol/L或VB_(12)0.1~2.0mmol/L均对氧化酪蛋白有保护作用;且随维生素添加量的增大,对氧化酪蛋白的保护效果增强。VC,VB_6,VB_2,VB_(12)量分别为8 mmol/L(0.28 g/g酪蛋白),2 mmol/L(0.082 g/g酪蛋白),0.3 mmol/L(0.023 g/g酪蛋白),0.6 mmol/L(0.162 g/g酪蛋白)时,对氧化酪蛋白的保护效果达最好。综上所述,酪蛋白氧化后发生蛋白交联和氨基酸功能基团变化,导致蛋白的变性程度增大及溶解度下降;VB_6,VB_2,VB_(12)均对氧化酪蛋白有良好的保护作用;而VC在低浓度时促进酪蛋白的氧化,高浓度时抑制酪蛋白的氧化。     

酒精对人原代培养肝细胞的氧化损伤与CYP 2E1关系的研究

目的 研究急性酒精暴露下对人原代培养肝细胞中CYP 2E1依赖的毒性作用和氧化损伤。方法 分离培养人原代肝细胞，以25-100mmol/L乙醇作用于人原代肝细胞9h及100mmol／L乙醇作用于人原代肝细胞0～24h后，检测人原代肝细胞中CYP 2E1的含量，并研100mmol/L乙醇作用于人原代肝细胞0～24h后，天冬氨酸转胺酶(aspartate transaminase，AST)的释放量及肝细胞中谷胱甘肽(Glutathione，GSH)、丙二醛(Malonclialdehyde，MDA)的含量。结果 急性酒精暴露导致人原代肝细胞中CYP 2E1的释放增加，并呈明显的剂量效应和时间效应关系；在100mmol/L乙醇作用下，AST和MDA明显升高，在0～24h内呈明显的时间效应关系，而GSH含量在6h后明显降低。结论 100mmol／L乙醇急性暴露可导致人原代培养肝细胞明显的氧化损伤，这种损伤与CYP 2E1活性的变化直接相关。     

高效液相色谱法对鹅肉中维生素E的含量的测定

目的:建立反相高效液相色谱-二极管阵列检测法(HPLC-DAD)测定动物性食品中维生素E(α-生育酚)的含量。方法:称取一定量鹅肉样品加入乙醇、氢氧化钾及抗坏血酸,100℃水浴皂化30min,乙醚提取后,减压浓缩,氮气吹致干,用脱醛乙醇溶解,进样10μL分析;色谱条件为:SunFireTmC18柱C18柱(15cm×4.6mm,5μm),流动相:甲醇∶水(98∶2),柱温30℃,检测波长300nm。结果:维生素E在0.10-50.0mg/L浓度范围内相关系数r=0.9985。结论:该方法能快速、灵敏、准确实现对动物性食品中脂溶性维生素E的含量分析。     

牡蛎寡肽对H2O2氧化损伤L02人肝细胞的保护作用

为了研究牡蛎寡肽对自由基清除作用及对人肝细胞L02细胞氧化损伤的保护作用,测定了牡蛎寡肽对羟自由基(·OH)的清除率,并建立过氧化氢(hydrogen peroxide,H_2O_2)氧化损伤人肝细胞L02的模型,研究牡蛎寡肽对氧化损伤L02细胞的存活率、活性氧(Reactive Oxygen Species,ROS)、抗氧化酶系(superoxide dismutase/SOD,glutathione/GSH)含量的影响。结果表明,牡蛎寡肽对羟自由基的IC_(50)为0.38 mg/m L。2 mg/m L牡蛎寡肽对于L02细胞的增殖率仍为123.98%。模型组SOD和GSH水平分别降低了40%、64.87%,而ROS增加了1倍。当添加不同剂量牡蛎寡肽后,中剂量组细胞中SOD含量几乎恢复到正常组水平,GSH活性也提高了118.89%,ROS水平降到模型组的62.43%,说明牡蛎寡肽对L02细胞无毒,能降低氧化损伤的L02细胞内的ROS水平,显著提高SOD和GSH含量,对细胞起到保护作用。因此,牡蛎寡肽对H2O2致氧化损伤的人肝L02细胞具有保护作用,可能是通过清除自由基,保护细胞内抗氧化酶系活性,抑制ROS的过多积累来实现。     

高效液相色谱法测定功能性食品中维生素C的含量

目的建立高效液相色谱法测定功能性食品中维生素C的含量。方法采用Venusil XBP C_(18)(L)柱(4.6mm×250 mm,5mm)为色谱柱,0.1%的草酸溶液为流动相,流速为0.5 m L/min,柱温为25℃,检测波长为245nm,样品经L-半胱氨酸-三乙胺溶液还原后,测定维生素C的含量,进样量20mL。结果维生素C在2.056~30.84mg/m L浓度范围内呈良好线性关系(r=0.9999),定量限为0.03726mg/m L,检出限为0.01242mg/m L,回收率为99.89%,RSD为0.31%(n=6);样品经L-半胱氨酸-三乙胺溶液还原后稳定性、重复性好。结论本方法简便快捷、适用范围广、准确度高、精密度好,可广泛用于功能性食品中维生素C含量的测定。     

鲍内脏多糖的抗氧化活性

为研究鲍内脏多糖的抗氧化活性,采用体外抗氧化实验,评价不同化学组成的鲍内脏多糖对1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基和·OH的清除作用,并以人体肝细胞LO2建立过氧化氢损伤模型,探讨鲍内脏多糖在细胞水平的抗氧化能力。结果表明:鲍内脏多糖CAVP、AVP1、AVP2具有良好的清除体外自由基能力。多糖CAVP、AVP1、AVP2清除DPPH自由基的半抑制率浓度(half maximal inhibitory concentration,IC50)分别为1.46、1.74、1.55 mg/m L。其清除·OH的IC50分别为7.14、15.27、8.11 mg/m L。另外,细胞模型法评价结果显示,鲍内脏多糖CAVP、AVP1、AVP2在质量浓度0.5~4.0 mg/m L条件下对肝细胞LO2的H2O2氧化损伤有保护作用,3种多糖样品均能显著提高LO2细胞存活率;CAVP(4.0 mg/m L)、AVP1(0.5 mg/m L)和AVP2(0.5 mg/m L)能极显著降低氧化损伤时乳酸脱氢酶的释放;CAVP能极显著提高LO2细胞内谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)(4.0 mg/m L)、过氧化氢酶(catalase,CAT)(0.5 mg/m L)、超氧化物歧化酶(superoxide dismutase,SOD)(0.5 mg/m L)活力;AVP1质量浓度为4.0 mg/m L时,能极显著提高LO2细胞内GSH-Px、SOD活力,显著提高CAT活力;AVP2分别在质量浓度0.5、4.0 mg/m L时极显著提高LO2细胞内CAT活力;在质量浓度为4.0 mg/m L时,3种多糖样品对降低细胞氧化损伤产生的丙二醛(malondialdehyde,MDA)均有显著作用。因此,鲍内脏多糖是一类潜在的抗氧化物,可用于此类功能食品的开发。     

丁香多酚细胞抗氧化活性的研究

目的：研究丁香多酚对由H2O2引起的人肝细胞RBL氧化损伤的保护作用。方法：采用H2O2诱导建立细胞氧化损伤模型,通过测定细胞存活率、细胞抗氧化酶活性、丙二醛等指标,分析探讨丁香多酚对细胞损伤的保护作用。结果：结果表明,100μmol/L H2O2孵育24h可显著诱导RBL细胞损伤,使细胞存活力下降到24.64%,细胞经不同浓度的丁香多酚（0.1、0.5、2、10mg/L）与H2O2共孵育后,特别是10mg/L丁香多酚可使细胞存活率达到59.18%。丁香多酚可通过提高SOD、CAT、GSH-Px酶活性,降低MDA含量,促进受损的RBL细胞修复。结论：丁香多酚对H2O2诱导氧化损伤的RBL细胞具有显著的保护作用,可作为食源性抗氧化剂。     

黑莓黄酮的自由基清除活性与防护肝细胞氧化损伤研究

基于超声辅助提取技术,采用单因素和正交试验优化黑莓黄酮的提取工艺。试验结果表明:黑莓中黄酮物质的最优提取条件是:温度70℃,提取时间150 min,乙醇体积分数80%,料液比1∶30 g/m L。在此条件下,黑莓黄酮的得率为2.23 mg/g,是优化前的2.01倍。在此基础上研究了黑莓黄酮的自由基清除活性,结果显示:黑莓黄酮的总抗氧化能力等价于14.89μg trolox,且黑莓黄酮清除DPPH自由基、ABTS自由基的IC50值分别为0.57mg/m L,0.49 mg/m L。进一步研究了黑莓黄酮对肝细胞氧化损伤的保护作用,结果表明过氧化氢(H2O2)能引起肝细胞LO2发生氧化损伤(细胞存活率为60.6%),80μg/m L的黑莓黄酮能显著抑制H2O2引发的细胞氧化损伤(细胞存活率为85.04%)。深入探究黑莓黄酮的作用机理发现,H2O2能引发肝细胞LO2中产生大量的活性氧自由基ROS(与正常对照组相比,荧光强度为276.62%);80μg/m L的黑莓黄酮能显著抑制H2O2引发的ROS(与正常对照组相比,荧光强度为139.19%),从而防护肝细胞的氧化损伤。     

固相萃取-高效液相色谱法测定婴幼儿配方粉中维生素A和维生素E的含量

目的建立固相萃取-高效液相色谱法同时测定婴幼儿配方粉中维生素A和维生素E的含量。方法样品经高温皂化反应后,将皂化液通过EXtrelut NT20固相萃取柱,对目标待测物进行净化,洗脱液经氮气吹干复溶后,过滤膜直接进样,以甲醇作为流动相,通过Zorbax C18色谱柱(4.6 mm×150 mm,5μm)分离,外标法定量。结果维生素A标准溶液在0.5~5.0 mg/L范围内,维生素E标准溶液在5.0~50 mg/L范围内均呈现良好的线性关系,相关系数(r~2)均≥0.999;样品3个水平加标回收率分别为86.0%~104.6%和89.0%~104.0%,相对标准偏差(RSD)均3.0%;检出限分别为1.0和10μg/100 g。结论该方法可用于婴幼儿配方粉中维生素A和维生素E的同时检测,操作简单、快速、准确。     

南极磷虾蛋白水解产物超滤组分的抗氧化活性评价

目的 对南极磷虾(Euphausia superba)蛋白水解产物超滤组分进行系统的抗氧化活性评价。方法 利用胃蛋白酶和胰蛋白酶制备南极磷虾蛋白水解物(antioxidant hydrolysate of Euphausia superba protein, EPAH), 利用超滤技术制备抗氧化组分。以自由基清除实验、脂质过氧化抑制实验、体外DNA氧化损伤保护实验以及氧化损伤张氏肝细胞(Chang liver)模型进行抗氧化组分的活性研究。结果 利用超滤技术将EPAH按照分子量(molecular weight, MW)分为三个肽组分EPAH-I(MW<3.5 KDa)、EPAH-II(3.5 KDa5 KDa)。其中, EPAH-I显示出强的DPPH自由基和羟基自由基清除活性、脂质过氧化抑制作用和质粒DNA的氧化损伤保护作用以及对H2O2氧化损伤的张氏肝细胞显示出较强的保护作用。在5.0 mg/mL浓度下, EPAH-I可将氧化损伤张氏肝细胞(Chang liver)的活力由模型组的(49.51±4.18 )%显著提升至(70.58±4.52)% (P<0.05); 超氧化物歧化酶和谷胱甘肽过氧化物酶活力分别提高至(176.34±10.92) U/mg prot和(35.83±2.55) U/mg prot, 活性氧自由基含量降至2.76±0.18%, 膜脂质氧化产物丙二醛的含量降至(5.75±0.16) nmol/mg prot。结论 本实验制备的南极磷虾抗氧化组分(EPAH-I)具有显著的生物活性, 可用于抗氧化相关功能产品的研发。     

茶多酚协同荷叶碱的抗氧化活性及其对结肠癌细胞的抑制作用

目的：探讨茶多酚协同荷叶碱的体外抗氧化活性及茶多酚单独对结肠癌细胞Caco-2的毒性作用。方法：采用二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）法、2,2’-联氮双（3-乙基苯并噻唑啉-6-磺酸）二铵盐（2,2’-azinobis（3-ethylbenzothi azoline-6-sulfonic acid）ammonium salt,ABTS）法检测茶多酚、荷叶碱及二者协同对DPPH自由基、ABTS＋•的清除作用;采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法检测茶多酚、荷叶碱及二者混合物对Cu2＋/H2O2体系诱导的牛血清白蛋白（bovine serum albumin,BSA）氧化损伤的保护作用;采用噻唑蓝（methyl thiazolyl tetrazolium,MTT）法,AO/EB、DAPI荧光染色法检测茶多酚对Caco-2细胞的毒性。结果：茶多酚、荷叶碱具有清除DPPH自由基、ABTS＋•及保护BSA蛋白氧化损伤的作用且呈剂量依赖性,两者之间存在显著的协同效应;1 mg/mL茶多酚作用24 h能显著降低Caco-2细胞活力,诱导Caco-2细胞发生凋亡。结论：茶多酚能有效地清除DPPH自由基、ABTS＋•,抑制蛋白氧化,且与其他活性成分具有协同效应;茶多酚能抑制Caco-2细胞的活力。     

The relationship between oxidative damage and vitamin E concentration in blood, milk, and liver tissue from vitamin E supplemented and nonsupplemented periparturient heifers

This study investigated the relationship between oxidative damage and the effect of vitamin E supplementation in blood, milk, and liver tissue in 16 periparturient heifers. The question is whether measurements of oxidative and vitamin E status in blood of a periparturient cow are representative of the total body, given that blood concentrations of both vitamin E and oxidative stress products change around this period. The daily vitamin E intake of the vitamin E-supplemented Holstein-Friesian heifers (n = 8) was 3,000 international units and was started 2 mo before calving; the control heifers (n = 8) were not supplemented. Oxidative damage was determined on the basis of malondialdehyde (MDA) concentrations. Blood was sampled 9 times before calving, on calving day, and twice after calving. Liver biopsies were taken at wk −5, −1, and 2 relative to calving day. Milk was obtained from all heifers immediately after calving, the first 2 milkings and on d 3, 7, and 14 at 0600 h. Serum and liver tissue were analyzed for vitamin E, cholesterol, and MDA; and milk samples were analyzed for vitamin E, MDA, fat, protein, and somatic cell count. The results showed that vitamin E supplements increased both absolute vitamin E concentrations and the ratio of vitamin E to cholesterol in blood and liver tissue. Absolute vitamin E concentration in milk tended to be greater in supplemented cows. Based on the increased MDA blood concentrations at calving, it seems that dairy heifers experience oxidative stress. The effect of vitamin E on MDA differs between the blood, liver, and mammary gland. Vitamin E supplementation could not prevent the increase in blood MDA at calving, but the significantly lower MDA blood concentrations of supplemented cows in the 2 wk after calving suggest that vitamin E has a role in recovery from parturition-related oxidative stress. Vitamin E supplementation reduced oxidative damage in liver, whereas no obvious effect was found on milk MDA concentrations. A strong relationship was found between blood and liver vitamin E and the ratio of vitamin E to cholesterol. Concentrations of MDA in blood and milk were also strongly related. The results show that the relationship between oxidative damage and vitamin E differs within blood, liver tissue, and milk. This implies that oxidative and vitamin E status calculated on the basis of blood values alone should be interpreted with caution and cannot be extrapolated to the whole animal.     

高效液相色谱法测定罗汉果中VC的含量

目的：建立HPLC 色谱法测定罗汉果中VC 含量的方法。采用5g/L 草酶对罗汉果浸提15min,对提取液中VC 进行检测,具体方法：采用SinoChrom ODS-BP(250mm × 4.6mm,5μm)分离;流动相为体积分数0.05% 磷酸溶液- 甲醇(98:2,V/V);流速1.0mL/min;采用二极管阵列多波长检测器确定VC 的最大吸收波长为242nm;测定温度25℃;进样量10μL。结果表明：VC 溶液0.08～0.50mg/mL 范围线性关系良好,相关系数r=1;回收率为99.3%,RSD 为1.93%。结论：该方法操作简便快速,精密度和稳定性好,适用于罗汉果VC 含量的测定。     

Influence of dietary vitamin C and selenium,alone and in combination,on the composition and oxidative stability of meat of broilers

Information on the combined effect of dietary vitamin C and Se on the composition and oxidative stability of meat of broilers is not available in the literature. In the present experiment, male broiler chickens were fed a maize–wheat–soya diet supplemented with vitamin C at 280 and 560 mg/kg of diet, and Se (sodium selenite or selenised yeast; Se) at 0.3 mg/kg for 5 weeks. After slaughter, samples of thigh meat were analysed. The supplementation of diets with vitamin C or Se increased the protein concentration of the meat at the expense of fat. Vitamin C supplementation increased the vitamin C content of the meat in a dose-dependent manner and decreased the vitamin A concentration in the meat of broilers fed diets with sodium selenite or without a Se supplement. In the meat of the broilers that were fed these diets, the vitamin C decreased the lipid oxidation in meat that was stored for 5 days. No sparing effect of vitamin C was apparent on the amount of vitamin E in the meat. Selenised yeast was more effective in the enrichment of meat with Se than was selenite. Both Se sources increased the activity of glutathione peroxidase and the oxidative stability of the meat.     

野生猕猴桃与维生素C对大鼠酒精性肝脑损伤的预防性保护作用

本文研究野生猕猴桃与维生素C对大鼠酒精肝脑损伤的预防性保护作用。将40只雄性SD大鼠随机分为4组:正常组、模型组、维生素C组及野生猕猴桃组。正常组给予等量生理盐水,其余组灌胃56°红星二锅头建立酒精肝脑损伤模型(第一周剂量为8 m L/kg,第二周为10 m L/kg),每天1次,同时正常组、模型组给予普通饲料,维生素C组给予维生素C强化饲料(按420 mg/kg添加维生素C),野生猕猴桃组给予按30%比例添加野生猕猴桃的饲料。2周后检测大鼠肝功能、肝脂质含量和氧化应激水平,HE染色观察肝和脑病理变化。结果表明,与模型组比较,野生猕猴桃与维生素C可显著降低酒精肝脑损伤大鼠的血清ALT、AST、TG、TC、LPO,血清及脑MDA,升高血清及脑SOD(p0.01),野生猕猴桃降低或升高指标更显著(p0.05)。因此,野生猕猴桃和维生素C对酒精肝脑损伤均具有预防性保护作用,前者效果更好。     

Alcohol metabolizing and antioxidant activities of complex herbal extracts from medicinal plants

The objective of this study was to evaluate the alcohol-metabolizing and antioxidative activities of complex herbal extract (CHE). The alcohol-metabolizing activity of CHE was evaluated by assessing alcohol dehydrogenase (ADH) activity, acetaldehyde dehydrogenase (ALDH) activity, and protective effect against alcohol induced damage in in vitro and in vivo models. In this study, CHE treatment significantly increased ADH and ALDH activities and reduced cell death in alcohol-induced liver cell damage. Moreover, it also significantly reduced the serum alcohol and acetaldehyde concentrations in alcoholfed rats. To define the effect of CHE on alcohol metabolism, its antioxidative activities were estimated by measuring radical scavenging activity, ferric-reducing antioxidant potential ability, and thiobarbituric acid reactive substance, and the results demonstrated a higher antioxidative capacity of CHE than the vitamin C control at a high dose (10 mg/mL). Therefore, this study suggests that CHE can be a potential natural resource for the management of ethanol-induced liver toxicity.     

山楂黄酮对BRL-3A肝细胞DNA损伤的修复作用及其机制

目的:探讨山楂黄酮对BRL-3A肝细胞DNA损伤的影响及其机制。方法:MTT法检测不同浓度的山楂黄酮对乙醇损伤肝细胞的影响;单细胞凝胶电泳分析细胞DNA的损伤程度并计算Olive尾距;试剂盒检测细胞内超氧化物歧化酶(SOD)、丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-Px)等生理生化指标;实时定量PCR检测基因SOD的表达变化。结果:0.2,0.4和0.8 mg/mL的山楂黄酮能够显著修复乙醇诱导BRL-3A肝细胞的DNA损伤,这种修复作用与细胞抗氧化指标SOD、MDA和GSH-Px的改善密切相关。结论:山楂黄酮能够通过抗氧化修复肝细胞的DNA损伤,山楂黄酮具备潜在的肝损伤治疗价值。     

Effects of Dietary Rape Seed Oil, Copper(II) Sulphate and Vitamin E on Drip Loss, Colour and Lipid Oxidation of Chilled Pork Chops Packed in Atmospheric Air or in a High Oxygen Atmosphere

The effect of addition of rapeseed oil (canola), CuSO(4) and vitamin E (all-rac-α-tocopheryl acetate) to pig diets on pork meat quality (lipid oxidation, colour and drip loss) was studied. Pigs were reared on ten different diets, either a control diet (no supplementation of rapeseed oil, CuSO(4) or vitamin E) or 6% rapeseed oil diets supplemented with CuSO(4) (0, 35 or 175mg/kg) and vitamin E (0, 100 or 200mg all-rac-α-tocopheryl acetate/kg). The natural content of vitamin E originating from feed ingredients amounted to 9-23mg vitamin E (α-tocopherol) per kg feed. Muscle vitamin E levels reflected the dietary intake and pigs fed the control diet had significantly lower levels than pigs fed rapeseed oil diets. The quality of fresh pork chops packed in air or in 80% O(2):20% CO(2) was followed during chill storage for 8 and 13 days, respectively. Colour, as measured by tristimulus colorimetry of pork chops packed in 80% oxygen atmosphere, was significantly improved with respect to redness when compared to chops packed in air, regardless of dietary treatment. The low vitamin E content in pigs fed the control feed significantly decreased a values and the oxidative stability of pork chops during chill storage compared to the other feeding groups. Packing of chops in a high-oxygen atmosphere increased lipid oxidation, especially in chops with low levels of vitamin E. Supplementation of rapeseed oil diets with 100 or 200mg vitamin E significantly decreased lipid oxidation of chill stored chops. Supplementation with CuSO(4) did not influence meat quality attributes (drip loss, colour stability and lipid oxidation) for any of the storage conditions.