CN109096368B - 一种同时具有抗氧化和护肝活性的多肽及编码该多肽的基因与其制备方法和应用 - Google Patents
一种同时具有抗氧化和护肝活性的多肽及编码该多肽的基因与其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种同时具有抗氧化和护肝活性的多肽及编码该多肽的基因与其制备方法和应用。多肽的氨基酸序列如下：Tyr‑Phe‑Leu‑Pro。本发明中合成肽的抗氧化能力通过氧自由基吸收能力实验（ORAC）评价，乙醇可诱导肝细胞（LO2）造成一定程度损伤，该LO2细胞损伤模型评价合成肽的护肝效果，结果表明合成肽可降低乙醇对LO2细胞的损伤作用，在预防和治疗酒精性肝病上有广泛的应用前景。
Description
技术领域
本发明涉及保健品和药物领域，具体涉及一种同时具有抗氧化和护肝活性的多肽及编码该多肽的基因与其制备方法和应用。
背景技术
随着生活水平的提高及膳食结构的改变,酒精性肝病(alcoholic liverdisease,ALD)的发病率逐年上升。酒精性脂肪肝(alcoholic fatty liver,AFL)是酒精性肝病的一种，是由于长期饮用酒精导致酒精性肝损伤，其主要原因是乙醇在肝细胞内代谢产生的毒性代谢产物及其引起的代谢紊乱，其可能会发展成酒精性肝炎、肝纤维化甚至是肝硬化。酒精性脂肪肝是酒精性肝病早期的最显著病理变化，现常以营养支持、降血脂、不饱和脂肪酸和磷脂类药物治疗，但治疗效果不佳且部分药物副作用明显，因此对其有明显治疗效果的药物及控制效果的保健品已成为国内外研究的热点。
AFL主要是由于酒精在细胞内代谢过程中生成乙醛有关，长期摄人酒精可以使肝细胞线粒体功能紊乱，使氧化乙醛的能力下降，而乙醇的氧化速度不变甚至提高，造成乙醛生成与降解不平衡，导致肝内乙醛含量增加，乙醛可与蛋白质形成复合物，对肝细胞产生损伤的作用，且该复合物作对于机体是异物，诱导机体自身免疫系统，加重肝细胞的损伤。肝微粒体乙醇氧化酶系统被激活后，产生的活性氧会直接对肝细胞造成氧化损伤。
活性肽在调控人的生长发育、免疫调节和新陈代谢中具有关键作用，其广泛地存在于各种生物体内，现也有为其特殊功效而人工合成的活性肽。活性肽具有主动吸收、吸收速度快、吸收完整、低耗等优点，并且它的生理功能优于氨基酸和蛋白质，所以具有一定功能的肽类食品是国际上研究的热点，日本、欧洲、美国都推出了各种具有特殊功能的食品和食品添加剂。在专利CN201110193662.1中，香川恭一等人通过对小鼠的动物实验发现多肽Trp-Thr-Gln-Arg(WTQR)抑制了血清中AST和ALT活性的上升，同时抑制了肝脂质的中性脂肪、总胆固醇和游离脂肪酸的积累，改善了肝机能；杨浩然等人发现的乳源免疫调节肽(Pro-Gly-Pro-Ile-Asn,PG-PIPN)在小鼠模型中，能显著降低加药组小鼠的血清AST和ALT含量，并且加药组小鼠的MDA含量降低、SOD和GSH-PX含量明显升高，说明该肽对肝细胞的保护作用与自由基的清除，提高机体抗氧化能力有关。以上说明活性肽对于ALD的防治具有较大的潜力。
发明内容
为了克服现有技术的缺点和不足，本发明的目的在于提供了一种同时具有抗氧化和护肝活性的多肽及编码该多肽的基因与其制备方法和应用。本发明提供的多肽，可以作为功能因子应用到护肝保健品和药品的开发上。
本发明的目的通过如下技术方案实现。
一种具有抗氧化活性的多肽，氨基酸序列为Tyr-Phe-Leu-Pro，或者YFLP，其中，Tyr或Y表示英文名称为Tyrosine，中文名称为酪氨酸的氨基酸的相应残基；Phe或F表示英文名称为Phenylalanine，中文名称为苯丙氨酸的氨基酸的相应残基；Leu或L表示英文名称为Leucine，中文名称为亮氨酸的氨基酸的相应残基；Pro或P表示英文名称为Proline，中文名称为脯氨酸的氨基酸的相应残基。
进一步的，所述多肽是具有抗氧化和护肝活性多肽的基因。其中，UAC或UAU为酪氨酸的氨基酸相应基因密码子，UUC或UUU为苯丙氨酸的氨基酸相应基因密码子，UUA、CUG、CUA或CUC为亮氨酸的氨基酸相应基因密码子，CCG、CCA、CCU或CCC为脯氨酸的氨基酸相应基因密码子。
本发明还提供了一种具有抗氧化活性的多肽在降低乙醇对LO2细胞的损伤作用中的应用。
进一步的，所述应用包括在制备预防和治疗酒精性肝病的功能性食品、保健品及药品中的应用。
进一步的，所述应用还包括基于预防和治疗酒精性肝病在药学上能够接受的载体或促血脑屏障穿越因子制备成丸剂、胶囊剂、片剂或注射制剂。
进一步的，发明所述的氨基酸序列采用标准Fmoc方案，选用2-chlorotritylchloride resin树脂进行固相合成。同时也可通过基因工程技术合成，将编码基因接入到载体中，再将载体转录到原核表达体系大肠杆菌中或真核表达体系酵母中进行表达，然后对目标多肽进行分离纯化，获得一种同时具备抗氧化和护肝活性的多肽。
进一步的，本发明首先通过氧自由基吸收能力实验(ORAC)检测了合成多肽的抗氧化活性，发现其供氢能力强，同时作用于乙醇诱导的肝细胞(LO2)损伤模型，发现其在预防和治疗酒精性肝病上有广泛应用前景。
与现有技术相比，本发明具有如下优点和有益效果：
本发明中合成肽的抗氧化能力通过氧自由基吸收能力实验(ORAC)评价，乙醇可诱导肝细胞(LO2)造成一定程度损伤，该LO2细胞损伤模型评价合成肽的护肝效果，结果表明合成肽可降低乙醇对LO2细胞的损伤作用，在预防和治疗酒精性肝病上有广泛的应用前景。
附图说明
图1为序列为YFLP的多肽分子结构式。
图2为多肽YFLP的HPLC图。
图3为多肽YFLP的ESI-MS图。
图4为多肽YFLP对乙醇诱导LO2细胞毒性的影响对比图(纵坐标为细胞存活率，横坐标为合成多肽浓度)。
图5为不同浓度下多肽对乙醇诱导LO2细胞毒性影响的对比图(纵坐标为细胞存活率)。
图6为多肽对乙醇诱导LO2细胞线粒体膜电位影响的对比图(横坐标为单体荧光强度，纵坐标为聚集体荧光强度)。
具体实施方式
本发明公开了一种多肽及其应用。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及产品已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法进行改动或适当变更与组合，来实现和应用本发明技术。
实施例1：多肽固相合成
1、树脂选型
(1)采用标准Fomc方案，选用0.0125mmol，2-chlorotrityl chloride resin树脂，按照氨基酸序列YFLP的序列特征，加入0.3mol第一个Fmoc保护氨基酸，将DCC和5％(质量分数)DMAP加入到反应器振荡反应，用NMP冲洗树脂除去多余保护氨基酸。
(2)选用0.0125mmol，Wang树脂，方法同上；
2、合成过程
采用标准Fomc方案，选用偶合率较高的2-chlorotrityl chloride resin树脂，按照氨基酸序列YFLP的序列特征，使肽链从C端逐个向N端延伸，各氨基酸的用量为0.1mol，加入0.3molFmoc保护氨基酸，每步缩合都加入HOBT活化保护氨基酸的羧基，每步缩合采用20％哌啶DMF溶液(15ml/g)，处理20min，去除Fmoc保护基。肽侧链合成后，将含有树脂的肽链加入到如下反应液中：二氯甲烷(99％)三氟乙酸(1％)(体积分数)，将肽链从树脂上切割下来。再次将多肽加入到反应液：三氟乙酸(94.5％)、酒石酸乙二胺(2.5％)、蒸馏水(2％)、TIS(1％)(体积分数)中反应2h，脱去侧链保护基。所合成的多肽经高效液相色谱仪纯化，纯度达到99％以上，并经ESI-MS鉴定结构(如表1、图2和图3所示)。
表1
实施例2：合成多肽的ORAC值测定
氧化自由基吸收能力(Oxygen radical absorbance capacity,ORAC)是一种基于氢原子转移机理评价抗氧化能力的方法，被认为接近生物体抗氧化功能的方法。ORAC反应在37℃的75mM磷酸盐缓冲溶液环境中进行，合成多肽(YFLP)、还原性谷胱甘肽(GSH)、荧光素和AAPH自由基均溶解在75mM的磷酸盐缓冲溶液，其中缓冲溶液的pH值为7.4，其终浓度分别为20mg/L，20mg/L，78nM和9.95mM。以Trolox作为标准抗氧化物，其终浓度梯度为0.5-4.2μM，并以GSH作为阳性对照。将合成肽、GSH分别与荧光素混合，并在37℃条件下保温20分钟，之后加入偶氮化合物AAPH，在激发波长485nm，发射波长538nm处检测荧光强度，检测时长为3h，获取荧光衰退曲线。抗氧化剂的氧自由基吸收能力ORAC值通过荧光衰退曲线的保护面积与标准抗氧化物质(Trolox)的保护面积相比得到。ORAC值以Trolox当量表达，结果如表2所示。
表2合成多肽YFLP、GSH的ORAC值
实施例3：合成多肽作用于乙醇诱导肝细胞损伤模型的护肝应用
取处于对数生长期的LO2细胞接种于96孔细胞板中，每孔5000个细胞，置于CO2培养箱中培养24h后，实验组加入合成多肽浓度分别为0.25、0.5、0.75、1、1.25mmol/L的培养液，空白对照组加等量的培养液，每组设3个平行，放回培养箱继续培养24h。每孔加20μLMTT溶液(5mg/mL)，于培养箱孵育4h，弃上清，每孔加入150μL DMSO，在490nm波长下测各孔的吸光值OD。计算不同乙醇浓度处理后各组细胞的存活率：
计算结果如图4所示。图4为多肽YFLP对乙醇诱导LO2细胞毒性的影响对比图，采用单因素ANOVA来检验合成肽与不含肽对照组统计学差异以及多肽不同剂量之间的统计学差异；其中：ns表示P>0.05；*#表示P<0.05。
实施例4：合成多肽作用于乙醇诱导肝细胞损伤模型的护肝应用
将处于对数生长期的LO2细胞按照5000个/孔密度种于96孔板，置于细胞培养箱中铺板24h后，使其重新贴壁。实验组加入浓度为0.5mmol/L的合成多肽培养液预孵育24h，模型组加入等量的培养液，孵育24h，弃上清，实验组加入同时含有0.5mmol/L的合成多肽和0.8mol/L乙醇的培养液，模型组加入0.8mol/L乙醇的培养液，继续培养24h，空白对照组始终用培养液培养。每孔加20μL MTT溶液(5mg/mL)，于培养箱孵育4h，弃上清，每孔加入150μLDMSO，在490nm波长下测各孔的吸光值OD。计算各组细胞的存活率(如图5所示)。
实施例5：合成多肽作用于乙醇诱导肝细胞损伤模型的护肝应用
将处于对数生长期的LO2细胞按照5000个/孔密度种于96孔板，置于细胞培养箱中铺板24h后，使其重新贴壁。实验组加入浓度为1mmol/L的合成多肽培养液预孵育24h，模型组加入等量的培养液，孵育24h，弃上清，实验组加入同时含有1mmol/L的合成多肽和0.8mol/L乙醇的培养液，模型组加入0.8mol/L乙醇的培养液，继续培养24h，空白对照组始终用培养液培养。每孔加20μL MTT溶液(5mg/mL)，于培养箱孵育4h，弃上清，每孔加入150μLDMSO，在490nm波长下测各孔的吸光值OD。计算各组细胞的存活率(如图5所示)。
实施例6：线粒体膜电位检测评价多肽对乙醇致LO2细胞的保护作用
取处于对数生长期的LO2细胞接种于12孔细胞板中，每孔50000个细胞，置于CO2培养箱中培养24h后，实验组1mmol/L的合成多肽预孵育24h，模型组加入等量的培养液，孵育24h，弃上清，实验组加入同时含有1mmol/L合成多肽和0.8mol/L乙醇的培养液，模型组加入0.8mol/L乙醇的培养液，继续培养24h，空白对照组始终用培养液培养。之后用胰酶消化并收集各孔细胞离心，加0.5ml JC-1染色工作液染色半小时，用PBS溶液洗2次，待测。JC-1是一种线粒体荧光探针，能够依赖电势能在线粒体基质聚合。在功能正常的线粒体中，线粒体跨膜电位较高，JC-1在电势能的作用下形成聚合物，发出橙红色荧光。当线粒体功能紊乱时，线粒体膜电位会下降甚至丧失，JC-1在线粒体基质中无法聚集，只能以单体形式分散于细胞中，呈现绿色荧光。可根据JC-1染色后的细胞荧光颜色变化来检测线粒体膜电位的变化，检测结果如图6所示。
合成多肽的高剂量组(1mM)对细胞模型进行干预后，与模型组相比，LO2细胞的细胞存活率显著提高(P<0.05)，而订剂量组(0.5mM)与模型组没有显著性差异。线粒体膜电位实验表明，合成多肽可保护乙醇诱导LO2细胞所导致的线粒体膜电位(m)下降，减缓由于乙醇造成线粒体功能紊乱影响。上述结果表明，本发明中的合成多肽在达到一定浓度时候，可以抑制乙醇诱导的LO2细胞存活率降低，具有护肝作用，可应用于制备预防和治疗酒精性肝病领域。
序列表
<110> 华南理工大学
<120> 一种同时具有抗氧化和护肝活性的多肽及编码该多肽的基因与其制备方法和应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4
<212> PRT
<213> 四肽(Tetrapeptide)
<400> 1
Tyr Phe Leu Pro
1
Claims (3)
1.一种同时具有抗氧化和护肝活性的多肽的应用，其特征在于：在制备预防和治疗酒精性肝病的药品中的应用；所述氨基酸序列为Tyr-Phe-Leu-Pro，如SEQ ID NO：1所示。
2.根据权利要求1所述的一种同时具有抗氧化和护肝活性的多肽的应用，其特征在于该多肽能降低乙醇对LO2细胞的损伤作用。
3.根据权利要求1所述的一种同时具有抗氧化和护肝活性的多肽的应用，其特征在于，所述预防和治疗酒精性肝病的药品包括在药学上能够接受的载体，所述药品的剂型为成丸剂、胶囊剂、片剂或注射制剂。
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