CN116270609B - 苯酞过氧化物在制备抗炎药物中的用途 - Google Patents
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Abstract
本发明公开了苯酞过氧化物在制备抗炎药物中的用途,苯酞过氧化物的结构式如式I所示:本发明提供的苯酞过氧化物可以有效抑制炎症介质一氧化氮的生成,在相同条件下优于阳性对照槲皮素的活性。本发明所公开的苯酞过氧化物还能够有效抑制炎症因子肿瘤坏死因子‑α和白细胞介素‑6的分泌,可以有效抑制促炎关键酶诱导型一氧化氮合酶和环氧化酶‑2的表达。本发明所公开的苯酞过氧化物表现出很好的抗炎特性,同时无细胞毒性,在制备抗炎药物领域具有很好的应用前景。
Description
技术领域
本发明属于苯酞过氧化物的医药新用途技术领域,具体涉及苯酞过氧化物在制备抗炎药物中的用途。
背景技术
炎症是机体的生理性自动防御反应,多因外部因素入侵机体或组织损伤而激活产生。在机体受到外界刺激时,机体释放大量的炎症分子参与机体炎症应答,造成炎症损伤,过度的炎症反应会导致多种疾病的发生。临床常用的抗炎药物有非甾体类抗炎药和甾体类抗炎药,然而这两类药物的副作用及不良反应较多,因此,寻找高效、低毒、低(无)成瘾性的抗炎药,一直是医药科学领域期盼解决的问题。抑制炎症介质和炎症因子的产生,如一氧化氮(nitric oxide,NO)、肿瘤坏死因子(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL,如IL-6)等,抑制促炎关键酶的表达,如诱导型一氧化氮合酶(induciblenitricoxide synthase,iNOS)、环氧化酶-2(cyclooxygenase-2,COX-2)等,是药物发挥抗炎作用的主要机制。
CN115160335A的专利文献公开了一种苯酞二聚体及其制备方法和应用,将川芎须根自然阴干、粉碎,加入乙醇水溶液,渗漉提取,提取液减压浓缩得浸膏;将浸膏加水分散,石油醚反复萃取,合并萃取液,减压回收溶剂得石油醚提取物;取提取物过硅胶柱进行石油醚/乙酸乙酯梯度洗脱,不同Rf值下,分别收集得Fr.1-5五个组分;将五部分分别提取,得外消旋混合物(±)-1及其光学纯体(+)-1b、(-)-1a,化合物2和化合物3。抗炎活性可见化合物对LPS诱导RAW264.7巨噬细胞NO生成均有明显抑制作用。该文献报道的外消旋体(±)-1及其光学纯体(+)-1b、光学纯体(-)-1a以及化合物2和3对LPS诱导RAW264.7巨噬细胞NO生成均有明显的抑制作用,然而上述抗炎活性较好的几种化合物均具有一定的细胞毒性,且公开的化合物中均不含有过氧键。
中药当归来源于伞形科植物当归Angelicasinensis(Oliv.)Diels的干燥根,苯酞类化合物是当归中一类重要的活性成分。为了探索当归中具有抗炎活性的苯酞化合物,本发明对其开展了研究,从当归中分离得到一种结构新颖的含有过氧键的苯酞过氧化物,它表现出较强的抗炎活性,该化合物在抗炎活性方面未见相关报道。
发明内容
本发明解决的技术问题是提供了苯酞过氧化物在制备抗炎药物中的用途。
本发明为解决上述技术问题采用如下技术方案,苯酞过氧化物在制备抗炎药物中的用途,所述苯酞过氧化物的结构式如式I所示:
进一步限定,所述抗炎药物为抑制炎症介质生成的药物,该炎症介质为一氧化氮。通过研究发现苯酞过氧化物I对炎症介质NO生成有显著的抑制作用,其IC50为4.98±0.94μM,明显强于阳性对照槲皮素的活性(IC50为26.06±2.28μM),且对NO生成呈剂量依赖性。
进一步限定,所述抗炎药物为抑制炎症因子分泌的药物,该炎症因子为TNF-α或/和IL-6。通过研究发现苯酞过氧化物I对炎症因子TNF-α和IL-6分泌有显著的抑制作用,且呈剂量依赖性。
进一步限定,所述抗炎药物为抑制促炎关键酶表达的药物,该促炎关键酶为iNOS或/和COX-2。通过研究发现苯酞过氧化物I对促炎关键酶为iNOS和COX-2的表达有显著抑制作用,且呈剂量依赖性。
本发明与现有技术相比具有以下优点和有益效果:本发明提供的苯酞过氧化物可以有效抑制炎症介质一氧化氮的生成,在相同条件下优于阳性对照槲皮素的活性。本发明所公开的苯酞过氧化物还能够有效抑制炎症因子肿瘤坏死因子-α和白细胞介素-6的分泌,可以有效抑制促炎关键酶诱导型一氧化氮合酶和环氧化酶-2的表达。本发明所公开的苯酞过氧化物表现出很好的抗炎特性,同时无细胞毒性,在制备抗炎药物领域具有很好的应用前景。
附图说明
图1为苯酞过氧化物I对炎症模型细胞释放炎症介质NO的影响图(###:与空白组相比P<0.001;***:与模型组比较P<0.001);
图2为苯酞过氧化物I对炎症模型细胞分泌炎症因子TNF-α影响图(###:与空白组相比P<0.001;**:与模型组比较P<0.01;***:与模型组比较P<0.001);
图3为苯酞过氧化物I对炎症模型细胞分泌炎症因子IL-6影响图(###:与空白组相比P<0.001;**:与模型组比较P<0.01;***:与模型组比较P<0.001);
图4为苯酞过氧化物I对炎症模型细胞中iNOS和COX-2蛋白表达影响的Westernblot图;
图5为苯酞过氧化物I对炎症模型细胞中iNOS蛋白表达影响的柱状图(###:与空白组相比P<0.001;*:与模型组比较P<0.05;**:与模型组比较P<0.01;***:与模型组比较P<0.001);
图6为苯酞过氧化物I对炎症模型细胞中COX-2蛋白表达影响的柱状图(###:与空白组相比P<0.001;*:与模型组比较P<0.05;***:与模型组比较P<0.001)。
具体实施方式
以下通过实施例对本发明的上述内容做进一步详细说明,但不应该将此理解为本发明上述主题的范围仅限于以下的实施例,凡基于本发明上述内容实现的技术均属于本发明的范围。
实施例1
苯酞过氧化物I的制备:
将干燥并粉碎后的当归18kg,用体积分数为95%的乙醇渗漉提取,减压浓缩提取液得到当归乙醇提取物2.1kg,将乙醇提取物悬浮于4L蒸馏水中,用10L二氯甲烷萃取,减压浓缩萃取液得到二氯甲烷萃取物293g。将二氯甲烷萃取物经常压硅胶柱色谱分离,以石油醚-乙酸乙酯溶剂系统在体积比为1:0~4:1的范围内梯度洗脱,经TLC鉴定合并相同组分得到初始目标组分Fr1~Fr16,将初始目标组分Fr9继续经常压硅胶柱色谱分离,以石油醚-丙酮溶剂系统在体积比为15:1~8:1范围内梯度洗脱,经TLC鉴定合并相同组分得到中间目标组分Fr9a~Fr9g,将中间目标组分Fr9f经葡聚糖凝胶柱色谱纯化(以二氯甲烷-甲醇溶剂系统体积比为1:1洗脱),得到苯酞过氧化物I(12mg)。
苯酞过氧化物I的波谱数据:HR-ESIMS m/z:467.2045[M+Na+];1H NMR(400MHz,acetone-d6):δH 1.62(1H,overlapped,Ha-4),1.79(1H,overlapped,Hb-4),1.50(1H,overlapped,Ha-5),2.17(1H,overlapped,Hb-5),4.94(1H,dt,J=4.7,1.1Hz,H-6),7.30(1H,d,J=6.1Hz,H-7),2.05(1H,overlapped,H-8),1.17(1H,overlapped,Ha-9),1.37(1H,overlapped,Hb-9),1.17(1H,overlapped,Ha-10),1.30(1H,overlapped,Hb-10),0.87(3H,t,J=7.3Hz,H-11),1.84(1H,overlapped,Ha-4'),2.21(1H,overlapped,Hb-4'),1.34(1H,overlapped,Ha-5'),1.99(1H,m,Hb-5'),2.93(1H,m,H-6'),7.81(1H,d,J=7.3Hz,H-7'),2.28(1H,overlapped,Ha-8'),2.46(1H,ddd,J=15.8,10.7,4.8Hz,Hb-8'),1.48(1H,overlapped,Ha-9'),1.58(1H,overlapped,Hb-9'),1.30(2H,overlapped,H-10'),0.86(3H,t,J=7.2Hz,H-11'),3.72(3H,s,1'-OCH3);13C NMR(100MHz,acetone-d6)δC 166.3(C-1),92.3(C-3),83.2(C-3a),23.6(C-4),24.8(C-5),72.9(C-6),133.4(C-7),137.3(C-7a),39.2(C-8),30.3(C-9),21.3(C-10),14.3(C-11),165.0(C-1'),210.5(C-3'),59.5(C-3a'),28.7(C-4'),17.4(C-5'),34.0(C-6'),153.8(C-7'),134.5(C-7a'),42.2(C-8'),27.8(C-9'),23.1(C-10'),14.5(C-11'),52.6(1'-OCH3)。
实施例2
苯酞过氧化物I对炎症模型细胞释放炎症介质NO的影响:
RAW264.7细胞按1.5×105个/孔接种于96孔板中,每孔细胞悬液100μL,置于37℃、体积分数5%CO2细胞培养箱中培养24h。设置空白组、模型组和加药组。空白组为正常培养的细胞;模型组加入5μL含有LPS的完全培养基(终浓度为1μg/mL);加药组加入5μL LPS(终浓度为1μg/mL)和5μL不同浓度药物,每组设置3个复孔,加药后孵育16h。吸取各组细胞上清液50μL于96孔板中,每孔分别加入Griess I液和Griess II液各50μL,置于摇床上震荡10min,在540nm下测定各组吸光度值,根据Griess试剂标准曲线计算NO含量,进一步根据抑制率计算IC50值。并采用MTT法测定化合物对RAW 264.7细胞的细胞毒性,在有效浓度下未发现细胞毒性。
苯酞过氧化物I抑制炎症介质NO生成的活性结果如下(表1):
表1苯酞过氧化物I对炎症介质NO生成的抑制活性
化合物 | IC50(μM) |
苯酞过氧化物I | 4.98±0.94 |
槲皮素 | 26.06±2.28 |
从表1可以看出,苯酞过氧化物I对炎症介质NO的生成有明显的抑制活性,远优于阳性对照槲皮素的活性。从图1可以看出,苯酞过氧化物I对炎症介质NO的生成抑制活性呈剂量依赖性。
实施例3
苯酞过氧化物I对炎症模型细胞分泌炎症因子TNF-α的影响:
RAW264.7细胞按1.5×105个/孔接种于96孔板中,每孔细胞悬液100μL,置于37℃、体积分数5%CO2细胞培养箱中培养24h。设置空白组、模型组和加药组。空白组为正常培养的细胞;模型组加入5μL含有LPS的完全培养基(终浓度为1μg/mL);加药组加入5μL LPS(终浓度为1μg/mL)和5μL不同浓度药物,每组设置3个复孔,加药后孵育16h。收集细胞上清液,按ELISA检测试剂盒说明书检测细胞上清液中TNF-α的水平。并采用MTT法测定化合物对RAW264.7细胞的细胞毒性,在有效浓度下未发现细胞毒性。
结果如图2所示,苯酞过氧化物I对炎症因子TNF-α的分泌表现出显著的抑制活性,并呈剂量依赖性。
实施例4
苯酞过氧化物I对炎症模型细胞分泌炎症因子IL-6的影响:
RAW264.7细胞按1.5×105个/孔接种于96孔板中,每孔细胞悬液100μL,置于37℃、体积分数5%CO2细胞培养箱中培养24h。设置空白组、模型组和加药组。空白组为正常培养的细胞;模型组加入5μL含有LPS的完全培养基(终浓度为1μg/mL);加药组加入5μL LPS(终浓度为1μg/mL)和5μL不同浓度药物,每组设置3个复孔,加药后孵育16h。收集细胞上清液,按ELISA检测试剂盒说明书检测细胞上清液中IL-6的水平。并采用MTT法测定化合物对RAW264.7细胞的细胞毒性,在有效浓度下未发现细胞毒性。
结果如图3所示,苯酞过氧化物I对炎症因子IL-6的分泌表现出显著的抑制活性,并呈剂量依赖性。
实施例5
苯酞过氧化物I对炎症模型细胞中iNOS和COX-2蛋白表达的影响:
RAW264.7细胞按3×106个/孔接种于6孔板中,每孔细胞悬液2mL,置于37℃、体积分数5%CO2细胞培养箱中培养24h,加入100μL LPS(终浓度为1μg/mL)和100μL不同浓度药物,同时设置空白组、模型组和药物组。加药后孵育16h,使用RIPA裂解液提取细胞总蛋白,BCA法进行蛋白定量,沸水浴高温变性后进行SDS-PAGE凝胶电泳,转移至PVDF膜,5wt%脱脂奶粉室温封闭2h,用含1wt%的聚山梨酯-20(吐温-20)Tris-HCI缓冲盐溶液(TBST)洗涤条带,然后分别加入稀释好的一抗iNOS、COX-2和β-actin,4℃孵育过夜,TBST洗涤条带3次,各10min,加入HRP标记二抗,室温孵育2h,TBST洗涤条带3次,各10min,然后进行化学发光显影,采用ImageJ软件分析目的蛋白和内参蛋白的灰度值。并采用MTT法测定化合物对RAW264.7细胞的细胞毒性,在有效浓度下未发现细胞毒性。
结果如图4~6所示,苯酞过氧化物I对促炎关键酶iNOS和COX-2的蛋白表达表现出显著的抑制作用,并呈剂量依赖性。
以上实施例描述了本发明的基本原理、主要特征及优点,本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明原理的范围下,本发明还会有各种变化和改进,这些变化和改进均落入本发明保护的范围内。
Claims (1)
1.苯酞过氧化物在制备抗炎药物中的用途,所述苯酞过氧化物的结构式如式I所示:
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