CN114716404A - 一类细胞色素p450 46a1酶抑制剂及其应用 - Google Patents

一类细胞色素p450 46a1酶抑制剂及其应用 Download PDF

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CN114716404A
CN114716404A CN202210210105.4A CN202210210105A CN114716404A CN 114716404 A CN114716404 A CN 114716404A CN 202210210105 A CN202210210105 A CN 202210210105A CN 114716404 A CN114716404 A CN 114716404A
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cytochrome
inhibitor
enzyme
cyp46a1
sesamol
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吴敬敬
杜杰
王长远
赵婷婷
孙慧君
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Dalian Medical University
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Abstract

本发明的目的在于提供一类细胞色素P450(CYP)46A1酶抑制剂及其应用，该抑制剂为一类含有亚甲二氧基苯结构的化合物，包括芝麻酚、芝麻素和/或芝麻林素。所述抑制剂可用于CYP 46A1酶的表型研究，包括鉴定CYP46A1是否参与特定内源性或外源性物质的代谢反应及其贡献率。体外活性测定发现这一类化合物抑制CYP46A1酶活性的IC50值在3.35～25.8微摩尔左右，均具有较强的抑制活性。提示该化合物在鉴定CYP 46A1参与内源性和外源性物质代谢贡献率的使用中具有良好的应用前景。

Description

一类细胞色素P450 46A1酶抑制剂及其应用
技术领域
本发明属医药技术领域，具体涉及一类细胞色素P450 46A1酶抑制剂及其应用。
背景技术
细胞色素P450 46A1酶(简称CYP46A1)是一种神经元专属的细胞色素P450酶，它主要分布于正常脑组织的神经元和星形胶质细胞中，少量分布于神经视网膜上(Proc NatlAcad Sci U S A,1999；96:7238)。CYP46A1参与脑内胆固醇氧化反应，将大脑内的胆固醇代谢为24-羟基胆固醇(24-OHC)，24-OHC在正常生理条件下进入体循环，是胆固醇消除和稳态稳定的主要途径(Current opinion in lipidology,2001；12:105)。目前为止，CYP46A1介导的代谢反应除了胆固醇-24-羟化反应外，还有睾酮-16β羟化反应(Front MolNeurosci.2020；13:568641.)。
芝麻酚(sesamol)，又名3,4-亚甲二氧基苯酚，多存在于芝麻籽粒、芝麻油和芝麻粕中，是芝麻油的重要的香气成分，也是芝麻油重要的品质稳定剂。芝麻酚具有非常强的抗氧化能力，常用于食品、医药的抗氧化剂，同时它更是合成抗高血压药物、心血管药物的重要的起始原料，同时也是农药胡椒基丁基醚的原料。
芝麻素(sesamin)，是一种含于芝麻籽中的天然成分，具有优良的抗氧化作用，农业上用作除虫菊酯的增效剂，临床观察对气管炎有一定疗效，可用于抗病毒、杀菌剂、抗氧化剂、杀虫增效剂、治疗气管炎、调节免疫力。
芝麻林素(sesamolin)，多存在于芝麻籽粒、芝麻油中，具有清除体内自由基、增强肝脏对四氯化碳和乙醇的解毒作用、抗艾滋病毒、抗肿瘤、保护神经中枢系统以及调节免疫等功能，能够作为增强机体健康的保健品添加剂，也可开发成防病治病的天然药物，还可作为保持食品风味的防腐剂。
发明内容
本发明的目的是提供一类细胞色素P450 46A1酶抑制剂，即芝麻酚、芝麻素和芝麻林素，其可用于细胞色素P450 46A1的表型研究，包括鉴定细胞色素P450 46A1在药物代谢反应中的参与情况和贡献率。
本发明技术方案如下：
一类新型细胞色素P450 46A1(CYP 46A1)酶抑制剂，该类抑制剂为一类含有亚甲二氧基苯结构的化合物，包括芝麻酚、芝麻素和/或芝麻林素，它们的结构如下所示：
Figure BDA0003530656550000021
本发明还提供了所述细胞色素P450 46A1酶抑制剂的应用，其特征在于：所述抑制剂可用于细胞色素P450酶系的体外代谢表型研究，主要用于鉴定细胞色素P450 46A1是否参与内源性或外源性物质的代谢反应及其贡献率。
本发明所述抑制剂还可用于体外抑制细胞色素P450 46A1介导的内源性或外源性物质的代谢反应，三种化合物抑制细胞色素P450 46A1介导睾酮16β-羟化反应的IC50值分别为25.8μM、6.02μM和3.35μM，Ki分别为18.7μM、6.87μM和4.02μM，芝麻酚的抑制类型为非竞争型，芝麻素和芝麻林素的抑制类型均为混合偏竞争型。
本发明提供的细胞色素P450 46A1酶抑制剂的优点如下：
(1)高效性：芝麻酚、芝麻素和芝麻林素可有效地抑制细胞色素P450 46A1介导的睾酮-16β羟化反应，IC50值分别为25.8μM、6.02μM和3.35μM。
(2)来源于药食同源食物芝麻籽，简单易得：芝麻酚、芝麻素和芝麻林素是芝麻籽中含量丰富的天然化学成分，可通过分离纯化获得。
附图说明
图1.芝麻系列化合物的化学结构式(式Ⅰ：芝麻酚，式Ⅱ：芝麻素和芝麻林素)；
图2.芝麻系列化合物对CYP46A1介导的睾酮16β-羟化反应抑制效应图；
图3.芝麻系列化合物对CYP46A1介导的睾酮16β-羟化反应的抑制曲线；
图4.芝麻系列化合物对CYP46A1介导的睾酮16β-羟化反应的抑制动力学；A,C,E为双倒数作图，B,D,F为Dixon作图；
图5.芝麻系列化合物对人源神经组织细胞介导的睾酮16β-羟化反应的抑制效应图。
具体实施方式
下列实施例是为了进一步说明本发明，而不是要限制其范围。
实施例1
芝麻酚、芝麻素和芝麻林素用于人重组CYP46A1酶的抑制活性评价
芝麻酚、芝麻素和芝麻林素对人重组CYP46A1酶活性的抑制作用，具体步骤如下：
(1)100微升体外代谢反应体系中，包含4mM MgCl2，CYP46A1单酶浓度0.5mg/ml，底物胆固醇的浓度为5μM，芝麻酚终浓度范围为5～200μM，芝麻素的终浓度范围为2～200μM，芝麻林素的终浓度范围为1～200μM，于37℃条件下预孵5分钟。
(2)于反应体系中加入10μl NADPH(终浓度1mM)起始反应。
(3)反应10分钟后，加入100μl包含卡马西平(内标)冰乙腈，剧烈震荡后终止反应。
(4)采用高速冷冻离心机，在20,000×g的条件下，高速离心上述体系20分钟后，取上清，进行LC-MS/MS检测分析。
芝麻酚、芝麻素和芝麻林素对人重组CYP46A1酶的抑制效应如图2所示，抑制曲线和抑制动力学分别如图3和图4所示。
实施例2
芝麻酚、芝麻素和芝麻林素对不同来源神经细胞中CYP46A1酶的抑制活性评价
芝麻酚、芝麻素和芝麻林素对人源神经组织细胞中CYP46A1酶的抑制活性作用，具体步骤如下：
(1)采用标准方法复苏各组织来源的细胞系，包括：CYP46A1转染细胞和SH-SY5Y(神经母细胞瘤细胞)，培养3天后，进行传代，并接种于12孔板中继续培养，细胞接种密度为1×106个/ml，每孔细胞悬液总体积为1ml。
(2)细胞继续培养24小时后，加入睾酮(终浓度10μM)进行CYP46A1酶活性测试，同时分别加入芝麻酚、芝麻素和芝麻林素，其终浓度分别为50μM,20μM,20μM(二甲基亚砜终体积小于0.1％，v/v)，于37℃的二氧化碳培养箱反应60分钟。
(3)反应60分钟后，取出500微升细胞培养上清液，加入等体积包含卡马西平(内标)的冰乙腈后充分涡旋混匀，采用高速冷冻离心机，在20,000×g的条件下，高速离心上述体系20分钟后，取上清，进行LC-MS/MS检测分析。
(4)吸除剩余培养基，加入200微升细胞裂解液裂解并收集细胞，采用Lowry方法进行细胞蛋白定量。
芝麻酚、芝麻素和芝麻林素对神经细胞介导的睾酮16β-羟化反应的抑制效应图如图5所示。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。

Claims (5)

1.一类细胞色素P450 46A1酶抑制剂，其特征在于，该抑制剂为一类含有亚甲二氧基苯结构的化合物，包括芝麻酚、芝麻素和/或芝麻林素，它们的结构式如下所示：
芝麻酚：
Figure FDA0003530656540000011
芝麻素：
Figure FDA0003530656540000012
芝麻林素：
Figure FDA0003530656540000013
2.一种权利要求1所述细胞色素P450 46A1酶抑制剂的应用，其特征在于：所述抑制剂用于细胞色素P450酶系的体外代谢表型研究。
3.按照权利要求2所述细胞色素P450 46A1酶抑制剂的应用，其特征在于：所述抑制剂用于体外鉴定细胞色素P450 46A1是否参与内源性或外源性物质代谢反应及其贡献率。
4.按照权利要求2所述细胞色素P450 46A1酶抑制剂的应用，其特征在于：所述抑制剂用于体外抑制细胞色素P450 46A1介导的内源性或外源性物质的代谢反应。
5.按照权利要求2所述细胞色素P450 46A1酶抑制剂的应用，其特征在于，所述抑制剂的使用浓度范围如下：
芝麻酚：10～200μM
芝麻素：2～40μM
芝麻林素：1～20μM。
CN202210210105.4A 2022-03-03 2022-03-03 一类细胞色素p450 46a1酶抑制剂及其应用 Pending CN114716404A (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105899083A (zh) * 2014-01-08 2016-08-24 Cj第制糖株式会社 芝麻油及其制备方法
CN107405329A (zh) * 2015-03-23 2017-11-28 三得利控股株式会社 昼夜节律改善用组合物
CN109890376A (zh) * 2016-10-27 2019-06-14 三得利控股株式会社 PGC-1α活化用组合物
CN110691517A (zh) * 2017-05-31 2020-01-14 株式会社皇后篮 用于制备食用油的方法及使用所述方法制备的食用油和食物

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105899083A (zh) * 2014-01-08 2016-08-24 Cj第制糖株式会社 芝麻油及其制备方法
CN107405329A (zh) * 2015-03-23 2017-11-28 三得利控股株式会社 昼夜节律改善用组合物
CN109890376A (zh) * 2016-10-27 2019-06-14 三得利控股株式会社 PGC-1α活化用组合物
CN110691517A (zh) * 2017-05-31 2020-01-14 株式会社皇后篮 用于制备食用油的方法及使用所述方法制备的食用油和食物

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