CN116693604A - 解酒肽 - Google Patents
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Abstract
本发明属于生物医药技术领域，具体提供了一种全新的活性解酒肽,该肽的序列如SEQ ID NO.1或NO.2所示；本发明还对应提供了含该肽的组合物，可以是药品、保健品或食品。本发明的肽分子量小、易于合成、安全无毒副作用，安全性和实用性良好。
Description
技术领域
本发明属于生物医药技术领域，具体涉及解酒活性肽及其应用。
背景技术
醉酒是酒精中毒的俗称，是指患者一次饮用大量酒精(乙醇)后发生的机体机能异常的状态，分为急、慢性醉酒。众所周知，乙醇主要是通过口腔、食管、胃、肠粘膜等吸收至组织器官，肝脏是酒精代谢的主要器官。在肝脏内，90％的乙醇被乙醇脱氢酶(ADH)氧化为乙醛，乙醛被乙醛脱氢酶(ALDH)氧化为乙酸，最后生成CO2和H2O排出体外。大量研究证明，酒精及其有害代谢产物在体内积累是造成酒精性肝损伤的主要根源。因此，加快酒精代谢、减少有害代谢产物在体内停留对于维持机体健康具有重要意义。目前，从天然产物中寻找安全有效的活性成分提高酒精代谢及缓解酒精性肝损伤是一个热点话题。
已有研究发现从植物类、水产类、畜禽类及蛋奶类等蛋白酶解物中得到了具有加速酒精代谢、缓解酒精性肝损伤的生物活性肽。微生物是生物活性资源大宝库，胞内含有丰富的活性蛋白，而外源蛋白质分子经胃肠消化后产生的小分子肽可能具有更强的生物效应，但乳酸菌源活性肽在促进酒精代谢及缓解酒精性肝损伤中作用并未见报道。本发明基于前期从乙醇诱导的乳酸菌差异蛋白的酶解物中筛选出两种提高ADH的活性肽，探究了其在酒精代谢中的用途。
发明内容
本发明的一种目的在于提供两种全新的活性解酒肽YRPI和YRPG，其氨基酸序列分别为Tyr-Arg-Pro-Ile和Tyr-Arg-Pro-Gly；
本发明的另一个目的在于进而提供该四肽用于解酒的应用；
相应地，本发明还提供了含该四肽的组合物，如为解酒类产品，可以是用于解酒的药物、保健品或食品。
进一步地，任一所述解酒，包括防治急慢性酒精中毒和/或酒精性肝损伤，可至少产生如下任一效果：
(1)能够加快体内酒精代谢；
(2)能够缩短乙醇及有害代谢产物在体内的停留时间；
(3)提高肝脏中ADH和ALDH酶的活性；
(4)提高血液中T-AOC、GSH和/或CAT的水平，降低血液中MDA水平；
(5)提高肝脏中T-AOC、GSH和/或CAT的水平，降低肝脏中MDA水平；
(6)降低血液中AST和/或ALT酶的含量；
优选地，所述的组合物为解酒类药物，可以是颗粒、胶囊、口服液等。
进一步地，所述的解酒类药物还包含本领域技术人员公知的药用辅料。
与现有技术相比，本发明具有以下积极有益效果：
本发明的四肽分子量小、易于合成、安全无毒副作用，安全性和实用性良好。与现有技术中已有的解酒剂等药物相比，原料简单易于制备，单独灌用四肽能达到很好的解酒效果，更加便于在药物、食品及保健品中的应用，更加易于其在解酒中广泛应用。
附图说明
图1为本发明所述四肽对ADH激活率的结果图；
图2为本发明所述四肽对小鼠体内乙醇浓度的结果图；
图3为本发明所述四肽对小鼠肝脏中乙醇代谢酶的结果图(A：肝脏中ADH活性，B：肝脏中ALDH活性)；
图4为本发明所述四肽对小鼠血清中抗氧化酶的结果图(A：小鼠血清中T-AOC的含量，B：小鼠血清中GSH的含量，C：小鼠血清中CAT的含量，D：小鼠血清中MDA的含量)；
图5为本发明所述四肽对小鼠肝脏中抗氧化酶的结果图(A：肝脏中T-AOC的含量，B：肝脏中GSH的含量，C：肝脏中CAT的含量，D：肝脏中MDA的含量)
图6为本发明所述四肽对小鼠肝损伤标记酶的影响(A：小鼠血清中AST活性，B：小鼠血清中ALT活性)；
具体实施方式
利用固相法化学合成本发明的四肽Tyr-Arg-Pro-Gly(SEQ ID NO.1)和Tyr-Arg-Pro-Ile(SEQ ID NO.2)，四肽来源于前期通过虚拟酶解乙醇诱导的乳酸菌差异蛋白，下面通过具体实施方式对本发明的技术方案进行更加清楚、完整的描述，以便于本领域技术人员对本发明的理解，但是并不用于对本发明保护范围的限制。
实施例1
体外测定四肽(YRPI和YRPG)对ADH活性的影响，测定方法如下：准备2支测定管，在测定管中加入150μL缓冲液(包括75μL 0.1mM PH 8.8的焦磷酸钠缓冲液，25μL 11.5％(v/v)的乙醇，50μL 27mM NAD+)，再分别加入YRPI和YRPG溶液(50μL，6mmol/L)，然后37℃孵育5min；温育后在各支测定管中立即加入50μL、0.2U/mL的ADH酶溶液并充分混匀，然后读取各溶液在340nm波长的吸光度变化，每间隔1min读取一次，连续测定5min。以加入蒸馏水的吸收值为空白组。计算相对酶活力，结果如图1所示。
图1结果表明，四肽YRPI和YRPG体外可提高ADH的激活率。
实施例2
本实施例通过动物实验进一步证明本发明所述四肽对小鼠酒精代谢的影响，具体实验如下：
(1)构建动物实验小鼠模型
取80只昆明小鼠(20-22g)，在动物房适应性喂养7天，然后随机分为5组：空白对照组(Control)、模型组(Model)、阳性对照组(Positive)、YRPI组和YRPG组。
空白组：每天按照小鼠体重灌胃生理盐水(30mg/Kg)，灌胃生理盐水后一次性灌胃生理盐水12ml/Kg；
模型组：每天按照小鼠体重灌胃生理盐水(30mg/Kg)，灌胃生理盐水后一次性灌胃56度白酒12ml/Kg；
阳性组：每天按照小鼠体重灌胃联苯双酯乳浊液(150mg/Kg)，灌胃联苯双酯乳独液后一次性灌胃56度白酒12ml/Kg；
YRPI组：每天按照小鼠体重灌胃四肽溶液(30mg/Kg)，灌胃YRPI活性肽后一次性灌胃56度白酒12ml/Kg。
YRPG组：每天按照小鼠体重灌胃四肽溶液(30mg/Kg)，灌胃YRPG活性肽后一次性灌胃56度白酒12ml/Kg。
上述实验组在第一天灌胃白酒(空白组灌胃生理盐水)2h后，进行眼球取血于枸橼酸钠抗凝剂中，混匀，4℃备用。利用气相色谱内标法检测血液中乙醇浓度，结果如图2所示；酒精摄入导致模型组小鼠体内的乙醇浓度在2小时内急剧上升至4.69mg/ml。与模型组相比，30mg/Kg的YRPI灌胃处理可使乙醇含量显著降低至2.51mg/mL，清除率达到46％；30mg/Kg的YRPG灌胃处理可使乙醇含量显著降低至2.32mg/mL，清除率达到51％，所述两种四肽直接降低了酒精对小鼠的毒性作用。
然后继续进行灌胃处理，按照上述的灌胃方法、连续灌胃7天，最后一次灌胃后2h，收集小鼠的血清和肝脏组织：取小鼠的血液样品在转速为4000rpm、温度为4℃的条件下离心10min，制备血清；肝脏用生理盐水制备成10％的肝匀浆，然后浆肝匀浆样品在转速为3500rpm、温度为4℃条件下离心10min，然后收集肝匀浆的上清液。对灌胃7天收集的血清及肝脏进行以下检测：
(2)测定小鼠血清和肝脏中乙醇代谢酶的活性
采用试剂盒(本领域技术人员公知方法)检测肝脏中ADH和ALDH的活性，结果如图3所示；小鼠在连续摄入乙醇7天后，模型组肝脏中ADH和ALDH活性显著降低。
检测结果证明，连续灌胃本发明所述的YRPI解酒肽7天，可以显著提高肝脏ADH(p＜0.001)和ALDH(p＜0.05)酶活性，从而提高了小鼠的酒精代谢能力；YRPG解酒肽也可以显著提高肝脏ADH(p＜0.001)和ALDH(p＜0.05)酶活性。YRPI和TRPG活性肽通过增强乙醇代谢酶活性从而加快乙醇代谢，减少乙醇代谢物及ROS对肝脏造成的损伤。
(3)测定小鼠血清中抗氧化酶活性及丙二醇(MDA)水平
基于上述收集的小鼠血清，采用试剂盒测定小鼠血清中总抗氧化水平(T-AOC)、谷胱甘肽(GSH)、过氧化氢酶(CAT)以及丙二醛(MDA)的水平，结果图4所示；图4结果连续灌胃7天YRPI活性肽可显著增强小鼠血清中T-AOC(p＜0.05)、GSH(p＜0.05)、CAT(p＜0.05)抗氧化酶的水平，显著降低了血清中MDA(p＜0.05)水平。图4结果同时表明连续灌胃7天YRPG肽显著增强了小鼠肝脏中T-AOC(p＜0.05)和GSH(p＜0.05)水平，降低肝脏中MDA(p＜0.05)的含量，减少了持续大量饮酒对小鼠造成的氧化应激损伤。
(4)测定小鼠肝脏的抗氧化酶活性及MDA水平
基于上述收集的小鼠肝脏，采用试剂盒测定小鼠肝脏上清液中总抗氧化水平(T-AOC)、谷胱甘肽(GSH)、过氧化氢酶(CAT)以及丙二醛(MDA)的水平，结果图5所示；图5结果连续灌胃7天YRPI活性肽可显著增强小鼠肝脏中T-AOC(p＜0.001)、GSH(p＜0.01)、CAT(p＜0.01)抗氧化酶的水平，显著降低了血清中MDA(p＜0.01)水平。图5结果同时表明连续灌胃7天YRPG肽显著增强了小鼠肝脏中T-AOC(p＜0.01)，GSH(p＜0.001)和CAT(p＜0.05)酶活性，降低肝脏中MDA(p＜0.01)的含量，减少了持续大量饮酒对小鼠肝脏造成的氧化应激损伤。
(5)测定小鼠血清中谷草转氨酶(AST)以及谷丙转氨酶(ALT)的活性
基于上述收集的小鼠血清，采用试剂盒(本领域技术人员公知的方法)测定小鼠血清中的AST以及ALT的活性，结果如图6所示。由图6结果可知，与模型组相比，连续灌胃7天四肽YRPI显著降低了血清中ALT(p＜0.05)的水平；连续灌胃7天四肽YRPG显著降低了血清中AST(p＜0.05)和ALT(p＜0.05)的含量。因此，整体来看，YRPI和YRPG对酒精诱导造成的肝细胞损伤具有保护作用。
Claims (7)
1.SEQ ID NO.1或SEQ ID NO.2所示多肽在制备解酒组合物中的应用。
2.如权利要求1所述的应用，其特征是，该解酒组合物用于防治急慢性醉酒和/或酒精性肝损伤。
3.如权利要求1所述的应用，其特征是，所述解酒组合物用于产生如下至少一种效果：
1）加快体内酒精代谢；
2）缩短酒精有害代谢产物在体内的停留时间；
3）降低血液中丙二醛、谷丙转氨酶和/或谷草转氨酶的含量；
4）降低肝脏中丙二醛或ROS氧自由基的水平；
5）提高血液中T-AOC、GSH和/或CAT的水平；
6）提高肝脏中T-AOC、GSH和/或CAT的水平；
7）提高肝脏中ADH和/或ALDH酶的活性。
4.如权利要求1所述的应用，其特征是，所述解酒组合物是用于解酒的药品、保健品或食品。
5.含SEQ ID NO.1或SEQ ID NO.2所示多肽的解酒组合物。
6.如权利要求5所述的解酒组合物，其特征是，所述解酒组合物是药品、保健品或食品。
7.SEQ ID NO. 2所示多肽。
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