鳕鱼皮胶原蛋白肽制备及其对肝细胞损伤的保护作用

目的:研究鳕鱼皮胶原蛋白肽的制备工艺及不同浓度的鳕鱼皮胶原蛋白多肽对经LPS诱导损伤的Chang Liver细胞的修复作用。方法:以料液比、加酶量、酶解时间和酶解温度为因素,MTT试验所得细胞OD值为指标,通过正交试验探讨鳕鱼皮胶原蛋白肽的最佳酶解工艺,并经超滤、阴离子交换、分子层析对其纯化。通过试剂盒法检测该纯化肽作用Chang Liver细胞后,细胞内抗氧化酶和细胞上清液中转氨酶变化情况;采用Hoechst 33258染色法和Annexin V-FITC/PI双标流式细胞术观察鳕鱼皮胶原蛋白肽作用损伤Chang Liver细胞后的细胞凋亡情况。结果:最优酶解条件:以胰蛋白酶为水解酶,酶解温度50℃,p H 6,加酶量2 000 U/g,酶解时间8 h,经纯化得到蛋白肽GM2-2-3;GM2-2-3作用经LPS诱导损伤的Chang Liver细胞后,细胞氧化损伤明显减轻,细胞内SOD、CAT、GSH-PX活性上升,MDA的含量下降,并呈剂量依赖性;细胞上清液中AST、ALT、ALP、γ-GT、LDH活性有所下降,且呈剂量依赖性;与损伤组细胞相比,Chang Liver细胞形态较舒展,细胞染色质出现明显的聚集及边集化,细胞核皱缩现象减少,早期凋亡率降低,早期凋亡细胞变少。     

黄线狭鳕鱼皮胶原蛋白肽对D- 半乳糖致衰老小鼠改善作用

为探究黄线狭鳕鱼皮胶原蛋白肽对D-半乳糖致衰老小鼠改善作用,通过建造D-半乳糖致衰老小鼠模型,以制备的黄线狭鳕鱼皮胶原蛋白肽为研究对象,选用低剂量（200 mg/kg）、中剂量（400 mg/kg）和高剂量（800 mg/kg）胶原蛋白肽灌胃D-半乳糖致衰老小鼠模型,对皮肤抗衰老作用进行研究。通过测定小鼠皮肤中的水分、胶原蛋白、羟脯氨酸（hydroxyproline,HYP）、透明质酸（hyaluronic acid,HA）含量、小鼠血清和皮肤中超氧化物歧化酶（superoxide dismutase,SOD）、过氧化氢酶（catalase,CAT）活性、丙二醛（malondialdehyde,MDA）含量和谷胱甘肽过氧化物酶（glutathione peroxidase,GSH-Px）活性以及对小鼠皮肤组织形态学的观察,评价黄线狭鳕鱼皮胶原蛋白肽抗衰老作用效果。结果表明,黄线狭鳕鱼皮胶原蛋白肽可以显著提高D-半乳糖致衰老小鼠皮肤的水分、胶原蛋白、HYP 和HA 的含量,其中高剂量胶原蛋白肽组的效果最显著。此外,黄线狭鳕鱼皮胶原蛋白肽能显著增加衰老小鼠血清和皮肤中SOD、CAT 活性、提升GSH-Px 活性,并有效抑制MDA 的产生。通过病理切片观察小鼠皮肤组织形态,发现黄线狭鳕鱼胶原蛋白肽能有效减轻因衰老引起的皮肤粗糙、松弛、胶原纤维减少、排列障碍等现象。综上,黄线狭鳕鱼皮胶原蛋白肽对D-半乳糖致衰老小鼠有明显改善作用,有效延缓皮肤的老化进程。     

罗非鱼皮胶原酶解物对HaCat细胞生长的影响

研究经不同商业酶水解后得到的罗非鱼皮胶原酶解物对人皮肤角质细胞HaCaT生长的影响,并对胶原酶 解物的抗氧化活性与促进细胞生长作用的关系进行探讨。结果表明：罗非鱼皮经中性蛋白酶（neutral enzyme, NE）、碱性蛋白酶、胰蛋白酶水解得到的酶解产物中,NE酶解物（neutral enzymatic hydrolysate,NEH）具有显著的 体外抗氧化活性和促进HaCaT细胞增殖的活性（P＜0.05）;NEH（1～5 ku）在质量浓度为25 mg/mL时对HaCaT细胞 的生长具有显著的促进作用（P＜0.05）,并在24 h内能显著促进细胞划痕处伤口愈合（愈合率（81.10±5.77）%, P＜0.05）。经氨基酸组成分析和凝胶色谱分析发现,NEH（1～5 ku）主要包含2 个肽组分,NEH（1～5 ku）的 抗氧化氨基酸苯丙氨酸、亮氨酸、酪氨酸、赖氨酸、精氨酸和组氨酸的质量分数要显著高于其他分子质量的肽 组分（P＜0.05）。研究认为,经NE水解得到胶原肽分子质量为1～5 ku组分对HaCaT细胞生长具有促进作用,这可 能与肽组分中抗氧化氨基酸质量分数较高有关。     

酶解法制备鲟鱼皮活性肽条件优化及抗氧化能力

为提高从鲟鱼皮中提取的生物活性肽含量,以鲟鱼皮胶原蛋白为主要材料,以蛋白酶酶解后的肽得率为 主要考察指标,在单因素试验基础上利用正交试验分析加酶量、酶解温度、酶解时间对鲟鱼皮活性肽得率的影 响,并优化酶解条件。在此基础上以新鲜猪肉为模型,考察鲟鱼皮活性肽溶液处理对猪肉组织抗氧化酶（总超氧 化物歧化酶（total-superoxide dismutase,T-SOD）、谷胱甘肽过氧化物酶（glutathione peroxidase,GSH-Px）、 谷胱甘肽S转移酶（glutathione S-transferase,GST））活力的影响。结果表明：酶解鲟鱼皮胶原蛋白的最适 酶为碱性蛋白酶,最佳工艺条件为加酶量12 000 U/g、酶解温度60 ℃、酶解时间4 h,在此条件下,肽得率为 （12.59±0.98）%;鲟鱼皮活性肽可以提高猪肉组织T-SOD、GSH-Px和GST活性,具有抗氧化功效。     

大口黑鲈鱼皮胶原蛋白肽的制备及抗氧化活性研究

目的：探究酶种类对胶原蛋白肽抗氧化活性的影响。方法：从大口黑鲈(Micropterus salmoides)鱼皮中提取胶原蛋白,采用碱性蛋白酶、中性蛋白酶、木瓜蛋白酶和胰蛋白酶酶解胶原蛋白,得到碱性蛋白酶酶解胶原蛋白肽(APP)、中性蛋白酶酶解胶原蛋白肽(NPP)、木瓜蛋白酶酶解胶原蛋白肽(PP)、胰蛋白酶酶解胶原蛋白肽(TP),比较其总抗氧化活性、还原活性、对DPPH·、·OH 和O-2·清除能力以及亚铁离子螯合能力和脂质氧化抑制活性。结果：4种蛋白酶酶解胶原蛋白肽都具有抗氧化活性,且抗氧化能力与胶原蛋白肽浓度呈正相关,其中APP的抗氧化活性最佳,总抗氧化活性和还原活性分别为1.725和0.958,对DPPH·和O-2·清除率分别为56.35%和38.41%。结论：碱性蛋白酶酶解的胶原蛋白肽抗氧化活性更佳。     

鳕鱼皮胶原活性多肽的制备研究

以鳕鱼皮为研究对象,研究不同蛋白酶对鳕鱼皮酶解效果的影响.实验以水解度为指标,通过正交试验确定胰蛋白酶、碱性蛋白酶、中性蛋白酶、木瓜蛋白酶的最佳酶解条件.采用试剂盒法对4种酶解液进行抗氧化活性测定.结果显示,碱性蛋白酶酶解液的总抗氧化能力最强,达到2.431 U/mgprot;中性蛋白酶酶解液清除超氧阴离子自由基的能力最强.     

鮟鱇鱼皮胶原蛋白肽的抗氧化活性

选取铁离子还原体系和3 种自由基体系为指标,研究鮟鱇鱼皮胶原蛋白肽体外清除自由基效果和抗氧化作用。采用超滤膜分离鮟鱇鱼皮胶原蛋白肽组分,筛选出体外清除•OH活性最好的组分,再建立小鼠衰老模型,将分离得到活性最好的组分采用灌胃法,测定小鼠皮肤中超氧化物歧化酶（superoxide dismutase,SOD）、过氧化氢酶（catalase,CAT）、谷胱甘肽过氧化物酶（glutathione peroxidase,GSH-Px）及丙二醛（malondialdehyde,MDA）和羟脯氨酸（hydroxyproline,Hyp）的含量,研究鮟鱇鱼皮胶原蛋白肽体内抗氧化和清除自由基作用。结果表明：鮟鱇鱼皮胶原蛋白肽具有较强的还原能力且对O－2•、DPPH自由基、•OH具有清除效果,质量浓度为10 mg/mL时对•OH、DPPH自由基、O－2•的清除率和对Fe3+ 的还原能力分别为70.48%、68.78%、42.53%和0.676;分子质量小于2 000 D的鮟鱇皮胶原蛋白肽组分对•OH具有较好的清除效果,IC50为15.7 mg/mL;剂量为100 mg/（kg•d）时,显著提高了皮肤中SOD、GSH-Px、CAT的活性,较模型组分别增加20.9%、41.3%和58.1%,且显著抑制MDA的形成。     

荔枝果壳原花青素对中波紫外线诱导HaCaT细胞氧化损伤的保护作用

为评价荔枝果壳原花青素对中波紫外线（ultraviolet B,UVB）（波长280～320 nm）诱导人永生化角质形成细胞（HaCaT）氧化损伤的保护作用。构建UVB辐射HaCaT细胞氧化损伤模型,研究荔枝果壳低聚原花青素（litchi pericarp oligomolymeric procyanidins,LPOPC）及从中分离鉴定的6 种单体化合物对HaCaT细胞氧化损伤的保护作用。实验分为对照组、UVB照射组、实验组（阳性对照对氨基苯甲酸（para-aminobenzoic acid,PABA）、LPOPC及6 种单体化合物）,以CCK-8法测定各组细胞活力,采用试剂盒检测各组细胞内活性氧自由基（reactive oxygen species,ROS）相对含量,超氧化物歧化酶（superoxide dismutase,SOD）、过氧化氢酶（catalase,CAT）、谷胱甘肽过氧化物酶（glutathione peroxidase,GSH-Px）活力,还原型谷胱甘肽（glutathione,GSH）及丙二醛（malondialdehyde,MDA）含量。结果表明：LPOPC及6 种单体化合物的干预处理均可明显提高UVB诱导氧化损伤的HaCaT细胞活力,减少细胞内ROS和MDA的生成,增加SOD、CAT、GSH-Px的活力和GSH水平。6 种单体化合物中,原花青素A2的保护作用最明显,与阳性药物PABA的效果相当。结论：LPOPC及6 种单体化合物均可通过增强细胞内抗氧化能力、抑制脂质过氧化来明显改善受损细胞氧化应激损伤,对UVB诱导氧化损伤的HaCaT细胞具有良好的保护作用,提示原花青素A2是荔枝果壳原花青素中对氧化应激损伤防护作用最强的活性组分。     

鳕鱼骨胶原蛋白肽的抗氧化活性

以鳕鱼骨为原料,采用热水提取法提取胶原蛋白,并采用中性蛋白酶、复合蛋白酶、酸性蛋白酶和胃蛋白酶分别进行酶解制备鳕鱼骨胶原蛋白肽。以对.OH、DPPH.和O2-.自由基的清除率作为评价指标,比较4种蛋白酶酶解制备的鳕鱼骨胶原蛋白肽的抗氧化活性。结果表明:4种蛋白酶制备的鳕鱼骨胶原蛋白肽对3种自由基均有一定的清除能力,且其清除能力随样品质量浓度的增大而增强;4种蛋白酶酶解制备的鳕鱼骨胶原蛋白肽对3种自由基的清除能力各不相同,其中对.OH的清除能力最强且有较好的量-效关系;当多肽质量浓度在4～10 mg/mL时,4种蛋白酶酶解胶原蛋白肽对DPPH.的清除能力优于对O2-.的清除能力。     

胶原蛋白多肽铬对四氧嘧啶损伤肝细胞的保护作用

研究了胶原蛋白多肽铬(CPCC)对四氧嘧啶损伤肝细胞HL-7702的保护作用,并探讨其作用机理.方法是将肝细胞HL-7702分为正常对照组、四氧嘧啶组、胶原蛋白多肽铬组及四氧嘧啶 胶原蛋白多肽铬组,分别检测细胞存活率、细胞胞内活性氧(ROS)、胞外超氧阴离子(O2.-)含量及超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活力、脂质过氧化物丙二醛(MDA)含量,并检测胶原蛋白多肽铬在过氧化氢(H2O2)模型组中的作用.结果表明胶原蛋白多肽铬处理使四氧嘧啶损伤肝细胞存活率增加,胞内ROS生成减少,O2.-含量、SOD与GSH-Px低偿性升高及MDA含量降低.结论是胶原蛋白多肽铬处理对四氧嘧啶损伤肝细胞具有一定的保护作用,其作用机制可能与三价铬抗氧化性能有关.     

Inhibitory effect of antioxidant peptides derived from Pinctada fucata protein on ultraviolet-induced photoaging in mice

The superposition of ultraviolet radiation induced (photodamage) aging process is photoaging. Antioxidant peptides derived from Pinctada fucata protein as raw materials were smeared the back of light-aging. The effect of antioxidant peptides on wrinkle generating, skin restoration capacity after pulled up and measure the active of the superoxide dismutase (SOD), catalase (CAT), hydroxyproline, and glutathione peroxidase (GSH-Px) and lipid peroxidation by kit and histological examination of UVB-irradiated skin that the mitotic index of basal cells, the thickness ratio of the epidermis and dermis, fibroblast count, density of collagen fibers and volume density of dermal microvasculature were studied. The result showed that antioxidant peptides had slowed down the progress of wrinkles formation and skin elasticity deceasing induced by acute irradiation of UVB. It also significantly controlled the speed of lipid peroxidation and the reduction of the activity of SOD, GSH-Px, hydroxyprodline and CAT. Histopathological studies showed that the derived antioxidant peptides could reduce the thickness ratio of the dermis and epidermis and increase the mitotic index of basal cells, fibroblast count, density of collagen fibers and volume density of dermal microvascular, these outcomes confirmed the protective role of antioxidant peptides in the process of photoaging.     

不同分子质量小麦胚芽多肽的体内抗氧化活性

研究不同分子质量小麦胚芽多肽的体内抗氧化活性。利用超滤和纳滤技术对通过复合酶法制备的小麦胚芽多肽进行分级分段和纯化。通过建立小白鼠的氧化损伤模型,将170只小鼠随机分成17组(正常对照组、模型对照组、不同分子质量以及不同剂量的小麦胚芽多肽实验组),除正常对照组外,其余各组进行腹部注射600mg/(kg·d)D-半乳糖建立氧化损伤模型,5种不同分子质量的多肽组灌胃低、中、高剂量的小麦胚芽多肽,实验周期40d,眼球取血处死小鼠,测定血清、肝脏中的超氧化物歧化酶(SOD)活性、谷胱甘肽过氧化物酶(GSH-Px)活性、丙二醛(MDA)含量和总抗氧化能力(T-AOC),研究不同分子质量小麦胚芽多肽的体内抗氧化活性。结果表明：小麦胚芽多肽能够使小鼠血清和肝脏中SOD、GSH-Px以及T-AOC的指标升高,降低小鼠血清和肝脏中脂质过氧化物的MDA含量,具有良好的抗氧化活性。总体来说,随着多肽质量浓度的升高,抗氧化活性也随之增强,分子质量在2000D以下的小麦胚芽多肽具有较强的抗氧化活性。     

Isolation and identification of antioxidative peptides from porcine collagen hydrolysate by consecutive chromatography and electrospray ionization–mass spectrometry

The porcine skin collagen was hydrolyzed by different protease treatments to obtain antioxidative peptides. The hydrolysate of collagen by cocktail mixture of protease bovine pancreas, protease Streptomyces and protease Bacillus spp. exhibited the highest antioxidant activities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, metal chelating and in a linoleic acid peroxidation system induced by Fe²⁺. And degree of hydrolysis highly affected the antioxidant properties of the hydrolysates. Four different peptides showing strong antioxidant activity were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration chromatography, ion-exchange chromatography and high-performance liquid chromatography. The molecular masses and amino acid sequences of the purified antioxidant peptides were determined using electrospray ionization (ESI) mass spectrometry. One of the antioxidative peptides, Gln-Gly-Ala-Arg, was then synthesized and the antioxidant activities measured using the aforementioned methods. The results confirmed the antioxidant activity of this peptide, and adds further support to its feasibility as a provider of natural antioxidants from porcine skin collagen protein.     

Study on effect of jellyfish collagen hydrolysate on anti-fatigue and anti-oxidation

Many bioactive peptides possess specific biological properties that make potential ingredients of health-promoting foods. Jellyfish, which is rich in collagen, has high nutritious and medicinal value. In this study, jellyfish collagen hydrolysate (JCH) were produced. The in vivo anti-fatigue activity and in vivo antioxidant activity of JCH were determined, respectively. Climbing endurance tests of mice were carried out after 6w of JCH administration and the blood lactate, BUN, hepatic glycogen and muscle glycogen of mice were determined in the anti-fatigue test. SOD, GSH-Px and MDA in serum and liver homogenate were determined after 6w of JCH administration. Results showed that the climbing time of Low, High and Positive group was increased compared with that of Control group. And administration with JCH could significantly reduce blood lactic and BUN levels and increase hepatic glycogen and muscle glycogen. MDA contents of serum and liver homogenate in the Aging Model had a significant higher level. And the mice in the Aging Model was showed a decreased level in GSH-Px activity of serum and SOD activity of liver homogenate compared with the other groups. JCH could significantly alleviate fatigue of the mice and had an anti-oxidative effect on aging mice, indicating that jellyfish is worthy of further study.     

一种新型抗氧化五肽的纯化、鉴定与表征

以黑鲨鱼皮为原料,利用蛋白酶酶解制备和分离纯化抗氧化多肽,并探究其抗氧化机理。以1,1-二苯基-2-苦肼基（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基清除率为主要指标,以水解度为辅助指标,优化酶解工艺条件。通过反相高效液相色谱和电喷雾四极杆飞行时间质谱分离纯化和鉴定氨基酸序列并探究其体外抗氧化活性。结果显示,最佳水解蛋白酶为碱性蛋白酶,最优酶解工艺为pH 8.0、酶添加量311 U/mL、底物质量分数3%、酶解温度45 ℃、酶解时间4.9 h,所得的酶解液DPPH自由基清除率为77.61%,水解度为14.21%。经反相高效液相色谱分离,得到组分F19的DPPH自由基清除能力最强。经鉴定,选择抗氧化活性高的总离子流色谱峰（保留时间为10.02 min左右）进行一级质谱和二级质谱分析,获得纯化的新型抗氧化五肽,其分子质量为461 Da,氨基酸序列结构为Pro-Gly-Gly-Thr-Met,其DPPH自由基清除率为75.01%（1.0 mg/mL）,氧自由基吸收能力（以Trolox当量计）为2490.01 μmol/g。新型五肽的Pro和Met在抗氧化中起到关键性作用。本研究为黑鲨鱼皮抗氧化五肽的抗氧化机理及其构效关系提供了一定理论依据。     

Enzymatic hydrolysis of Alaska pollack (Theragra chalcogramma) skin and antioxidant activity of the resulting hydrolysate

BACKGROUND: Fish skin, a by‐product of the food industry, contains a large amount of collagen. However, only a small proportion of fish skin is used in the production of leather materials and animal feedstuffs, most of it being discarded. The aims of this study were to prepare peptides from Alaska pollack (Theragra chalcogramma) skin by enzymatic hydrolysis and to evaluate the antioxidant activity of the resulting hydrolysate. RESULTS: Protamex was the most efficient enzyme for preparing antioxidant peptides from Alaska pollack skin. The optimal hydrolysis conditions were as follows: hydrolysis time 8 h; enzyme/substrate ratio 2:1000; skin/water ratio 1:6; temperature 55 °C; pH 6.0. Under these conditions the highest yield of peptides was 83.44%, with 85.95% of the hydrolysate being mainly composed of oligopeptides with molecular weights ranging from 180 to 1000 Da. The hydrolysate showed 2,2‐diphenyl‐1‐picrylhydrazyl radical‐scavenging activity, with an IC₅₀ value of 2.5 mg mL^?1, and its reducing power was 0.14 at 1 mg mL^?1, 53.8% of that of reduced glutathione at the same concentration. CONCLUSION: This study demonstrated that the hydrolysate of Alaska pollack skin was mainly composed of oligopeptides with two to eight amino acid residues and possessed antioxidant activity. Copyright © 2010 Society of Chemical Industry     

Effects of Collagen and Collagen Hydrolysate from Jellyfish (Rhopilema esculentum) on Mice Skin Photoaging Induced by UV Irradiation

ABSTRACT:  Collagen (JC) was extracted from jellyfish ( Rhopilema esculentum ) and hydrolyzed to prepare collagen hydrolysate (JCH). The protective effects of JC and JCH against UV-induced damages to mice skin were evaluated and compared in this article. JC and JCH could alleviate the UV-induced abnormal changes of antioxidative indicators, including the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities and the contents of glutathione (GSH) and malondiaidehyde (MDA). JC and JCH could protect skin lipid and collagen from the UV radiation damages. Furthermore, the changes of total ceramide and glycosaminoglycan in skin were recovered significantly by JC and JCH. The action mechanisms mainly involved the antioxidative properties and the repairing to endogenous collagen synthesis of JC and JCH  in vivo . JCH with the lower molecular weight showed much higher effects than JC. The results indicated that JCH was a novel antiphotoaging agent from natural resources.     

哈蟆油非水溶性成分及其酶解物的抗氧化功效研究

研究哈蟆油非水溶性成分及其酶解物的抗氧化作用。采用D-半乳糖法建立小鼠过氧化损伤模型。设立哈蟆油非水溶性成分及其酶解物高、中、低(0.30、0.15、0.075g/kg)3个剂量组和模型对照组、空白对照组,灌胃给药30d。观察过氧化损伤模型小鼠肝组织中丙二醛(MDA)含量、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活力的变化。哈蟆油非水溶性成分可以显著降低肝组织中MDA含量,但对SOD、GSH-Px活力的升高无影响;哈蟆油非水溶性成分酶解物可显著降低丙二醛(MDA)含量,显著增加肝组织GSH-Px、SOD的活力。哈蟆油非水溶成分酶解物具有明显的抗氧化作用。