CN111686099A - 茶氨酸在制备缓解酒精性肝损伤产品中的应用 - Google Patents
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Abstract
本发明属于茶叶中的活性成分应用领域，具体涉及一种茶氨酸在制备缓解酒精性肝损伤药物/食品中的应用。本发明所要重点保护的是茶氨酸在制备缓解酒精性肝损伤药物/食品中的应用，尤其是茶氨酸在制备缓解急性酒精性肝损伤药物/食品中的应用。本发明所涉及的茶氨酸，主要是L‑茶氨酸。本发明通过大量的实验证实了茶氨酸对一次大量饮酒引起的急性肝损伤具有显著地缓解作用，并且明确了茶氨酸是通过NF‑κB相关的炎症因子信号通路的调控而发挥其缓解作用的。本发明提供的茶氨酸尤其是L‑茶氨酸在缓解急性酒精性肝损伤中具有显著效果；本发明的茶氨酸，是来源于茶叶的纯天然的原料，安全性高、绿色，对人体无副作用。
Description
技术领域
本发明属于茶叶中的活性成分应用领域，具体涉及一种茶氨酸在制备缓解酒精性肝损伤药物/食品中的应用。
背景技术
肝脏是酒精代谢的主要场所，饮酒后，乙醇在胃及小肠上段吸收入血，约98％乙醇在肝脏内代谢，若长期过量饮酒，使乙醇在体内不能及时代谢而蓄积，可对肝脏造成不同程度的损伤。酒精性肝损伤属于临床中常见的肝脏疾病，是由于长期饮酒而导致的肝脏损伤疾病，包括酒精性脂肪肝、酒精性肝炎、酒精性肝纤维化、酒精性肝硬化，这几种形式可以单独存在也可以同时存在。从组织学分析，酒对肝器官的损伤可表现为肝脏水肿、肝脂肪化以及不同程度的肝纤维化改变等；从生化指标的变化看，可有标志肝细胞线粒体损伤的谷草转氨酶(AST)升高、标志肝细胞膜损伤的谷丙转氨酶(ALT)升高。
CN107412665A公开了一种具有解酒、防治酒精性肝损伤的中药组合物及其制备方法和应用。本发明按重量份包括以下原料药制得：西洋参10-200份、枳椇子10-200份、生晒参10-200份、红景天10-200份、茯苓10-100份、贯众10-60份、陈皮10-60份、白蔻仁10-60份、补骨脂10-100份、甘草10-100份、隔山消10-200份、生山楂10-200份、高良姜10-200份、北柴胡10-200份、远志10-80份、石斛10-100份、生黄芩10-200份、猪苓10-200份、乌梅10-200份、野葛根10-200份、冬虫夏草10-200份、白芨10-200份、鸡矢藤10-200份、北五味子10-60份、刺五加10-100份。
上述的专利其成分较多，成本也相应的较高。
CN110840950A披露了俄色茶和/或俄色茶提取物在制备防治非酒精性肝病和/或非酒精性肝损伤的药品中的应用。俄色茶和/或俄色茶提取物对防治非酒精性肝病具备显著的防治作用。通过应用发现，俄色茶提取物能够有效地抑制肝系数的增加，从而抑制非酒精性脂肪肝的产生；俄色茶能够有效的抑制长期高脂饮食所造成的脂肪在肝细胞中的积累。
上述专利文献中并未明确具体在防治非酒精性肝病或者非酒精性脂肪肝的药品中起关键作用的具体物质；并且上述文献中起关键作用的物质其分子机制也并不明确。俄色茶和/或俄色茶提取物在酒精性肝损伤上是否具有功效也不明确。
发明内容
本发明通过大量的数据，验证了茶氨酸对一次大量饮酒引起的急性肝损伤具有显著地缓解作用，明确茶氨酸通过NF-κB相关的炎症因子信号通路的调控而发挥其缓解作用的。
茶氨酸在制备缓解酒精性肝损伤产品中的应用、茶氨酸在制备缓解酒精性肝损伤药物/食品中的应用，是本发明所要保护的重点。
尤其的，上述的酒精性肝损伤为急性酒精性肝损伤。
上述的茶氨酸主要是指L-茶氨酸。
包括含有上述茶氨酸的能缓解酒精性肝损伤的药物/食品，也是本发明所要保护的范围。
L-茶氨酸的用量为100～400mg/kg/d。
上述的药物中，L-茶氨酸是唯一活性原料，其它原料是常规药用辅料。
上述的L-茶氨酸占药物重量的15～30％，余量为常规药用辅料。
L-茶氨酸是通过NF-κB相关的炎症因子信号通路的调控而发挥其缓解作用的。
更具体的，L-茶氨酸通过调控TNF-a/NF-κB信号通路来发挥其在制备缓解急性酒精性肝损伤药物中的作用。
上述的药物其制剂为注射液、皮下埋植剂、片剂、粉剂、颗粒剂、胶囊、口服液或缓释剂中的任一种。上述的食品为固体食品或液体食品；优选的，所述的食品为液体饮料、粉状食品、膏状食品、块状食品中的任一种。
茶氨酸是一种主要在茶科植物中存在的独特的氨基酸，在绿茶、乌龙茶、红茶中均存在,含量依茶树品种和加工方式的不同有较大变化，在绿茶中含量较高，是绿茶鲜爽滋味的主要来源。茶氨酸一般占茶叶干物质总重的1-2％。茶氨酸不参与蛋白质的合成,以游离形式存在，占茶叶中游离氨基酸总量的40％-70％。自然界存在的茶氨酸为L型，系统命名为：N-乙基-L-谷氨酰胺，极易溶于水，可通过小肠壁吸收。研究表明茶氨酸作为茶叶中主要的功能成分之一具有多种健康功效，如抗肿瘤、抗菌、免疫调节、抗抑郁、缓解神经疼痛等。
关于茶氨酸有应用，江春柳等人在《茶氨酸的制备及应用研究进展》一文中，披露了茶氨酸具有抗肿瘤的作用、降压的作用、对神经系统也有重要作用，在食品中用作功能性食品添加剂，主要是用于使人放松和提高记忆力，还可以作为茶饮料的品质改良剂、风味改良品剂、以及净水剂净化饮用水和除臭剂等。但是关于将其应用于缓解急性酒精性肝损伤的治疗药物或食品中的文献，鲜有披露。
和背景技术中的专利文献相比，本发明明确了茶氨酸尤其是L-茶氨酸对一次大量饮酒引起的急性肝损伤具有显著地缓解作用，并进一步明确茶氨酸通过NF-κB相关的炎症因子信号通路的调控而发挥其缓解作用的。
本发明解决了以下的问题：
(1)一次大量饮酒引起的肝脏损伤是临床上常见的肝脏疾病，严重威胁着人类健康，寻找能够缓解急性酒精性肝损伤的治疗药物及功能成分物质是亟待解决的问题，而本发明提供了茶氨酸尤其是L-茶氨酸在解决上述问题上具有显著效果，为缓解急性酒精性肝损伤的治疗药物或缓解急性酒精性肝损伤的食品提供了一种安全的原料；
(2)本发明所提供的茶氨酸，其是来源于植物(茶叶)的纯天然的原料，安全性高、绿色，对人体无副作用；
(3)茶氨酸能够显著缓解小鼠一次性大量饮酒引起的肝损伤程度，显著降低大量饮酒后小鼠血清中AST及ALT水平，并能够升高小鼠肝脏中超氧化物歧化酶SOD及谷胱甘肽过氧化物酶的水平，降低肝损伤小鼠肝脏中丙二醛的水平。
本发明对茶氨酸缓解小鼠急性酒精性肝损伤的分子机制进行研究，结果显示，茶氨酸通过调控TNF-α/NF-κB信号通路，从而抑制NF-κB介导的炎症反应，抑制iNOS、IL-1β、IL-6等炎症因子的表达，进而缓解酒精引起的急性肝损伤作用。本发明通过实验和大量数据研究，表明了茶叶中的功能成分物质——茶氨酸是一种具有明显的缓解急性酒精性肝损伤的活性物质。
附图说明
图1为L-茶氨酸对酒精性柑损伤小鼠肝脏损伤的缓解作用；
图2为L-茶氨酸对急性酒精性肝损伤小鼠血清中酶活性的影响作用；
图3为L-茶氨酸对小鼠肝脏抗氧化活性的影响作用；
图4为L-茶氨酸调控小鼠肝脏中TNF-α/NF-κB信号通路及通路相关炎症因子的表达作用。
具体实施方式
下面结合具体实施例对本发明作更进一步的说明，以便本领域的技术人员更了解本发明，但并不因此限制本发明。
实施例1
本发明对小鼠进行一次性大量饮酒制作小鼠肝损伤模型，比较给茶氨酸组小鼠与对照组给相同剂量的无菌水组的小鼠肝损伤程度，小鼠肝脏病例切片比较显示，茶氨酸显著的缓解了小鼠一次性大量饮酒引起的肝损伤程度，明显的降低大量饮酒后小鼠血清中AST及ALT水平，并能够升高小鼠肝脏中超氧化物歧化酶SOD及谷胱甘肽过氧化物酶的水平，降低肝损伤小鼠肝脏中丙二醛的水平。
同时，本发明人对茶氨酸缓解小鼠急性酒精性肝损伤的分子机制进行研究，结果表明，茶氨酸通过调控TNF-α/NF-κB信号通路，从而抑制NF-κB介导的炎症反应，抑制iNOS、IL-1β、IL-6等炎症因子的表达，进而缓解酒精引起的急性肝损伤作用。这说明，本发明专利显示茶叶中的功能成分物质——茶氨酸是一种具有明显的缓解急性酒精性肝损伤的活性物质。
本发明的研究，关于L-茶氨酸在缓解急性酒精性肝损伤作用中的实验，其具体的步骤如下：
S1：L-茶氨酸(纯度>98％)购自Sigma-Aldrich，实验中试剂盒购自南京建成公司，western bloting所用的抗体购自CST公司；
S2：SPF级雄性C57BL/6J小鼠60只，购自北京维通利华实验动物技术有限公司，体重为25±2g。饲养环境条件为20～25℃，相对湿度50～70％。实验小鼠在实验开始前在标准SPF级动物房中适应一周，自由取食饮水。
适应结束后，将实验小鼠随机分成5组，每组12只：正常组、模型组、L-茶氨酸低剂量组(灌胃100mg/kg BW的L-茶氨酸)、L-茶氨酸中剂量组(灌胃200mg/kg BW的L-茶氨酸)、L-茶氨酸高剂量组(灌胃400mg/kg BW的L-茶氨酸)。各组小鼠禁食12h后，除正常组外，均以5ml/Kg BW的无水乙醇灌胃，正常组灌胃等剂量的蒸馏水，30min后各治疗组分别给相应剂量的L-茶氨酸，模型组和正常组灌胃等体积生理盐水。给药2h后用高浓度二氧化碳处死小鼠，并取血。对小鼠进行心脏灌注清除掉器官残存的血液。解剖小鼠，取肝脏，分为三部分，一部分用4％多聚甲醛固定，用于HE染色等分析；一部分放置于-80℃冰箱保存用于后续western bloting实验，剩余的部分按1：10加入预冷的1.15％氯化钾溶液分别制成肝组织和胃组织匀浆，匀浆液放置于-80℃冰箱保存。上述解剖所取血液4℃静置2h以上，3000rpm离心10min，吸取上清液，移至EP管中，封口，置-80℃冰箱中保存，用于后续实验；
S3：小鼠肝脏病理切片：将解剖的小鼠肝脏组织在10％的甲醛溶液中保存24h，石蜡包埋，切片，苏木精、伊红染色，在显微镜下观察并拍照；
S4：生化指标检测：取小鼠血清，用试剂盒对不同处理的小鼠血清中AST及ALT的活性进行检测；
S5：Western blot免疫印迹法分析L-茶氨酸缓解急性酒精性肝损伤的机制取小鼠肝脏组织，加入含PMSF的裂解液，组织匀浆机中进行破碎，于冰中静置30min，然后在4℃，18506rcf离心20min。将上清液小心移入预冷的1.5ml EP管中。BCA法进行组织总蛋白测定后，进行SDS电泳。并转移到PVDF膜(微孔)。用5％干脱脂奶将膜封住过夜，然后在室温下与一抗孵育1小时(1:1000稀释)。膜免疫印迹有以下主要抗体:鼠标单克隆抗体针对IL-1β,il–6，进气阀打开，IKKa TNF-αNF-κB和p-NF-κB(Ser536)。用含0.05％ten-20的trs缓冲盐水(TBST)冲洗后，与辣根过氧化物酶标记的二抗(1:3000稀释)在室温下孵育1小时。用ECL增强发光液孵育PVDF膜，室温作用2分钟。将膜置于Canon凝胶成像系统中显影。
S6：数据分析
所有数据均以平均值±标准差(x±SD)表示，使用Graph Pad Prism 7.0软件进行数据分析。在三种茶样之间进行one-way ANOVA分析，同行或同图中不同小写字母表示在0.05水平上差异显著。
实施例2
(1)茶氨酸对小鼠酒精性肝损伤的缓解作用
各组小鼠肝脏组织分别用苏木精和伊红染色。如图1所示，与正常对照组(CG)肝脏组织相比，酒精处理模型组(MG)肝细胞凋亡明显，肝组织充血、肿胀、空泡、颗粒变性。而与模型组相比，L-茶氨酸处理组肝脏组织切片的组织学和结构形态学均有改善，特别是高剂量处理组肝脏组织接近正常组。
(2)L-茶氨酸对小鼠血清中酶的活性的影响作用
ALT与AST活性及两者的比值是肝损伤，肝脏炎症的重要的标志性指标，本发明人对小鼠血清中ALT与AST的活性进行检测，结果显示(图2)，与对照组相比，模型组血清ALT、AST活性明显升高。相比之下，与模型组相比，使用100、200和400mg/kg的L-茶氨酸处理显著降低了两种酶的活性，同时，L-茶氨酸处理显著降低了AST/AST比值。因此，血清生化检验支持组织学检查的结果。
(3)L-茶氨酸对小鼠肝脏组织中抗氧化水平的影响作用
肝脏中抗氧化酶的水平是肝脏健康水平的一个重要指标，本发明人检测了各组小鼠肝脏组织中超氧化物歧化酶(SOD)、丙二醛(MAD)、谷胱甘肽过氧化物酶(GSH-Px)的水平，结果如图3所示，急性酒精性肝损伤小鼠肝脏中SOD及GSH-Px水平显著降低，而L-茶氨酸处理后能够显著增加肝脏中SOD及GSH-Px的水平。肝脏中丙二醛(MAD)是肝脏损伤的重要指标，本发明人的检测结果显示，肝损伤模型小鼠肝脏中MAD显著升高，L-茶氨酸处理显著降低了小鼠肝脏中MAD的水平。附图中的结果表明，L-茶氨酸具有显著的缓解急性酒精性肝损伤的作用。
(4)L-茶氨酸缓解急性酒精性肝损伤作用的分子机制
用western blotting对小鼠肝脏中炎症相关的蛋白表达水平，结果显示(图4)，与正常组小鼠相比，酒精性肝损伤的小鼠肝脏中TNF-a及IKKa的表达显著升高，L-茶氨酸处理的小鼠肝脏中TNF-a、IKKa表达水平显著降低。NF-κB与生物体内的炎症反应密切相关。因此，本发明人检测了不同处理小鼠肝脏中NF-κB及其磷酸化水平，结果显示，急性酒精性肝损伤小鼠肝脏中NF-κB磷酸化水平显著升高，表明急性酒精性肝损伤可能是通过NF-κB的激活而介导肝脏产生一系列的炎症反应的产生。同时，本发明人对iNOS、IL-1β及IL-6等炎症因子进行了检测，结果显示，急性肝损伤模型小鼠的肝脏中炎症因子的表达水平显著升高，L-茶氨酸处理能够显著降低上述炎症因子的表达水平。因此，上述结果表明，L-茶氨酸是通过调控TNF-a/NF-κB信号通路来发挥其缓解急性酒精性肝损伤作用的。
通过以上的实验，表明了茶叶中的生物活性物质——L-茶氨酸能够显著缓解一次大量饮酒引起的小鼠急性酒精性肝损伤的水平，具有明显的缓解急性酒精性肝损伤的作用。
对L-茶氨酸缓解急性酒精性肝损伤作用的分子机制进行了探索，结果显示，L-茶氨酸能够通过调控炎症反应相关的TNF-α/NF-κB信号通路及通路相关炎症因子的表达水平来发挥其缓解急性酒精性肝损伤的功效。
Claims (10)
1.茶氨酸在制备缓解酒精性肝损伤产品中的应用。
2.茶氨酸在制备缓解酒精性肝损伤药物/食品中的应用。
3.如权利要求1所述的茶氨酸在制备缓解酒精性肝损伤药物/食品中的应用，其特征在于，所述的酒精性肝损伤为急性酒精性肝损伤。
4.如权利要求1所述的茶氨酸在制备缓解酒精性肝损伤药物/食品中的应用，其特征在于，所述的茶氨酸为L-茶氨酸。
5.含有茶氨酸的具有缓解酒精性肝损伤作用的药物/食品；
优选的，含有L-茶氨酸的具有缓解酒精性肝损伤作用的药物/食品。
6.如权利要求1所述的茶氨酸在制备缓解酒精性肝损伤药物/食品中的应用，其特征在于，所述的L-茶氨酸的用量为100～400mg/kg/d。
7.如权利要求1所述的茶氨酸在制备缓解酒精性肝损伤药物/食品中的应用，其特征在于，所述的药物中，L-茶氨酸是唯一活性原料，其它原料是常规药用辅料；
所述的食品中，L-茶氨酸是唯一活性原料，其它原料是常规食品辅料或者是常规食品添加剂。
8.如权利要求1所述的茶氨酸在制备缓解酒精性肝损伤药物/食品中的应用，其特征在于，所述的药物中，L-茶氨酸占药物重量的15～30％，余量为常规药用辅料；
所述的食品中，L-茶氨酸占食品重量的15～30％，余量为常规食用辅料。
9.如权利要求1所述的茶氨酸在制备缓解酒精性肝损伤药物/食品中的应用，其特征在于，L-茶氨酸是通过NF-κB相关的炎症因子信号通路的调控而发挥其缓解作用的；
优选的，L-茶氨酸通过调控TNF-a/NF-κB信号通路来发挥其在制备缓解急性酒精性肝损伤药物/食品中的作用。
10.如权利要求1所述的茶氨酸在制备缓解酒精性肝损伤药物/食品中的应用，其特征在于，所述的药物其制剂为注射液、皮下埋植剂、片剂、粉剂、颗粒剂、胶囊、口服液或缓释剂中的任一种；
所述的食品为固体食品或液体食品；
优选的，所述的食品为液体饮料、粉状食品、膏状食品、块状食品中的任一种。
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| AU2021101179A AU2021101179A4 (en) | 2020-07-17 | 2021-03-05 | Application of theanine in preparation of products for alleviating alcoholic liver injury |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101953825A (zh) * | 2010-09-25 | 2011-01-26 | 万红贵 | 一种解酒组合物及其在制备保健品中的应用 |
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| CN101953825A (zh) * | 2010-09-25 | 2011-01-26 | 万红贵 | 一种解酒组合物及其在制备保健品中的应用 |
Non-Patent Citations (4)
| Title |
|---|
| JE PEREZ-VARGAS等: "L-Theanine prevents carbon tetrachloride-induced liver fibrosis via inhibition of nuclear factor κB and down-regulation of transforming growth factor β and connective tissue growth factor", 《HUMAN & EXPERIMENTAL TOXICOLOGY》 * |
| 中国中医药年鉴（学术卷）》编辑委员会: "《中国中医药年鉴 2018 学术卷》", 31 December 2018, 上海辞书出版社 * |
| 叶寅: "L-茶氨酸通过增强肝脏细胞中抗氧化物质活性抑制酒精性肝损伤的研究", 《中国优秀博硕士学位论文全文数据库（硕士）医药卫生科技辑》 * |
| 贲培玲等: "L-茶氨酸生物制备技术和生物功能研究进展", 《中国茶叶加工》 * |
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