CN111269230A - 蜡梅碱-2,3二酮及其制备方法和医药用途 - Google Patents
蜡梅碱-2,3二酮及其制备方法和医药用途 Download PDFInfo
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Abstract
本发明公开了从山蜡梅的干燥叶中分离得到的一种新的生物碱化合物蜡梅碱‑2,3二酮,具有如下结构:
Description
技术领域
本发明属于药物领域,具体涉及从山蜡梅的干燥叶中分离得到的一种新的三生物碱类化合物蜡梅碱-2,3二酮及其制备方法和医药用途。
背景技术
甲型流感病毒H1N1是人类最常感染的流感病毒之一,易变异、传染性强、传播快、发病率和死亡率高。从世纪以来已出现过数次全球性的大流行,由于我国人口基数大,密集程度大,流行性感冒在我国传播更加迅猛,给我国人民的健康带来巨大隐患。
目前,治疗甲型流感病毒的药物主要有两大类,分别是以离子通道蛋白为靶点的金刚烷胺和金刚乙胺以及以神经氨酸酶为靶点的扎那米韦和奥司他韦。由于流感病毒是分节段的病毒,容易发生变异并容易对现有药物产生耐药性,如欧洲已经出现了对金刚烷胺及奥司他韦产生耐药性的工毒株。此外,现有的抗流感病毒药物大多具有不同程度的毒副作用,尤其是离子通道阻断剂具有神经毒性等副作用。因此,研发低毒有效的抗甲型流感的药物十分迫切。
山蜡梅作为江西的道地药材,根据七七版《中国药典》记载山蜡梅叶具有解表祛风,清热解毒,其在预防感冒、流行性感冒等方面有较好疗效。在江西婺源地区,民间常自制山蜡梅茶(又称毛山茶)用于防治感冒,并具有较好疗效。随着现代科学技术发展,人民对蜡梅属植物进行现代植物学研究,发现蜡梅属植物中主要含有萜类、黄酮类、生物碱类、挥发油类、香豆素类等物质,这些化学物质的活性逐渐被发现,现在已广泛应用于各个领域。虽然蜡梅属植物中化学成分在各领域的作用都有报道,但其在抗流感病毒方面的却鲜有报道。
发明内容
本发明的目的是提供一种从山蜡梅中分离出的具有抗甲型流感病毒H1N1作用的新的生物碱化合物蜡梅碱-2,3二酮。
具有下述结构式的化合物
本发明所述的化合物,可以与酸形成药学上可成的盐。可以为有机酸盐或无机酸盐,如盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硝酸盐、磷酸盐、乙酸盐,苯甲酸盐,马来酸盐,富马酸盐,苹果酸盐,柠檬酸盐,草酸盐,乳酸盐,琥珀酸盐,酒石酸盐,烷基磺酸盐、芳基磺酸盐,氨基酸盐(如半胱氨酸盐)等。
本发明的另一个目的在于提供上述化合物的制备方法,包括如下步骤:
(1)取山蜡梅叶,用水和/或醇提取,得提取物;
(2)将步骤(1)所得提取物进行酸提,过滤得酸水部分;
(3)将步骤(2)所得酸水部分用碱调节pH至7-12,使用三氯甲烷萃取,取三氯甲烷部位;
(4)将步骤(3)所得三氯甲烷部分加硅胶H拌样,装柱,进行硅胶柱色谱分离,依次采用体积比300:1、300:2、300:4、300:8、300:16、300:32、300:64、300:128、300:250、300:500、300:1000的二氯甲烷-乙醇溶液各洗脱1-3个柱体积(优选2个柱体积),后再用甲醇洗脱1-5个柱体积(优选3个柱体积),通过薄层硅胶色谱TCL检测、合并相同的馏分,依次得到7个馏分;
(5)取步骤(4)所得的第2馏分,通过凝胶柱色谱分离,优选葡聚糖凝胶色谱柱,更优选Sephadex LH-20,采用甲醇溶液洗脱1-5个柱体积(优选3个柱体积),依次得到两个馏分,取第2馏分,经半制备高效液相色谱得到权利要求1所述化合物,C18反相色谱柱,流动相为体积比为40:60的甲醇-水溶液,检测波长230nm。
本发明一个具体的示例,采用半制备HPLC(waters-2489),ODS柱(5μm,i.d.20mm×250mm,YMC Co.)进行色谱分离,流速为9mL/min。本发明所述化合物的保留时间为37min。
优选的,步骤(1)采用体积百分比为80%-95%的乙醇水溶液进行提取。
优选的,步骤(2)采用盐酸进行酸提。所述酸的质量体积百分比为2%。
优选的,步骤(3)使用浓氨水调节pH。优选pH=9。
优选的,可对步骤(1)、(2)、(3)获得的产物进行浓缩用于下一步制备。
本发明一个具体的制备方法,包含以下操作步骤:
(1)取干燥后的山蜡梅叶,放入提取罐内,用浓度为95%,87%,80%乙醇分别提取三次,每次50分钟,合并、浓缩三次提取液,回收乙醇,得总浸膏。
(2)取总浸膏,用温水将其分散,用2%的盐酸对其进行“酸提”,分多次进行提取,直至浸膏中生物碱反应为阴性,得溶于酸水部分和不溶于酸水部分(非生物碱部位),过滤酸水部分和不溶于酸水部分,取酸水部分的生物碱浸膏。
(3)用浓氨水调节酸水部位的浸膏至pH=9,用三氯甲烷萃取3-4次,直至生物碱显色反应为阴性,回收三氯甲烷,得pH=9三氯甲烷部位的浸膏。
(4)取pH=9的生物碱浸膏,用50mL甲醇充分溶解,加硅胶(100~200目)拌样,硅胶H装柱,进行硅胶柱色谱分离,依次采用体积比300:1、300:2、300:4、300:8、300:16、300:32、300:64、300:128、300:250、300:500、300:1000的二氯甲烷-乙醇溶液各洗脱2个柱体积,后再用甲醇洗脱3个柱体积,通过薄层硅胶色谱TCL检测合并相同的馏分,得7个馏分,称重并编号为A1、A2、A3、A4、A5、A6、A7。
(5)A2馏分的分离纯化:取A2馏分通过凝胶柱色谱Sephadex LH-20(i.d.3cm×120cm)分离,采用甲醇溶液洗脱3个柱体积,得A2-1~2两个馏分,取A2-1馏分,经半制备高效液相色谱,HPLC(waters-2489),ODS柱(5μm,i.d.20mm×250mm,YMC Co.),流动相为体积比为40:60的甲醇-水溶液,检测波长230nm,流速为9mL/min,进行色谱分离。本发明所述化合物的保留时间为37min。
本发明的另一目的在于提供本发明化合物在制备抗流感病毒,特别是流感病毒H1N1药物中的应用。本发明所述化合物具有抑制甲型流感病毒H1N1的作用。
体外实验表明,蜡梅碱-2,3二酮具有很好的抗流感病毒H1N1活性。采用CellTiter-GloTM试剂盒检测该化合物对细胞的毒性作用、采用测定流感病毒蛋白表达水平来检测病毒的复制水平的方法检测该化合物的抗甲型流感病毒活性,药物作用于甲型流感病毒H1N1的EC50为29.92μg/ml,CC50为331μg/ml。用利巴韦林作阳性对照。结果表明蜡梅碱-2,3二酮可以用于制备抗甲型流感病毒H1N1的药物。
附图说明
图1为本发明所述化合物的1H-NMR谱图。
图2为本发明所述化合物的13C-NMR谱图。
图3为本发明所述化合物的HSQC谱图。
图4为本发明所述化合物的HMBC谱图。
图5为本发明所述化合物的HMBC相关简图。
图6为本发明所述化合物绝对构型确定。
图7为本发明所述化合物对H1N1的抑制作用和对MDCK细胞的毒性作用的曲线图(图A为细胞存活率,图B为细胞抑制率)。
具体实施方式
下面通过对本发明实施例的描述,详细说明但不限制本发明。
实施例1化合物的制备
取干燥后的山蜡梅叶,放入提取罐内,用浓度为95%,87%,80%乙醇分别提取三次,每次50分钟,合并、浓缩三次提取液,回收乙醇,得总浸膏。取总浸膏,用温水将其分散,用2%的盐酸对其进行“酸提”,分多次进行提取,直至浸膏中生物碱反应为阴性,得溶于酸水部分和不溶于酸水部分(非生物碱部位),过滤酸水部分和不溶于酸水部分,取酸水部分的生物碱浸膏,用碱对其进行“碱沉”,用浓氨水调节酸水部位的浸膏至pH=9,用三氯甲烷萃取3-4次,直至生物碱显色反应为阴性,回收三氯甲烷,得pH=9三氯甲烷部位的浸膏。
取pH=9的生物碱浸膏,用50mL甲醇充分溶解,加硅胶(100~200目)拌样,硅胶H装柱,进行硅胶柱色谱分离,依次采用体积比300:1、300:2、300:4、300:8、300:16、300:32、300:64、300:128、300:250、300:500、300:1000的二氯甲烷-乙醇溶液各洗脱2个柱体积,后再用甲醇洗脱3个柱体积,通过薄层硅胶色谱TCL检测合并相同的馏分,得7个馏分,称重并编号为A1、A2、A3、A4、A5、A6、A7。取A2馏分通过凝胶柱色谱Sephadex LH-20(i.d.3cm×120cm)分离,采用甲醇溶液洗脱3个柱体积,得A2-1~2两个馏分,取A2-1馏分,经半制备高效液相色谱,HPLC(waters-2489),ODS柱(5μm,i.d.20mm×250mm,YMC Co.),流动相为体积比为40:60的甲醇-水溶液,流速为9mL/min,检测波长230nm,进行色谱分离,本发明所述化合物的保留时间为37min.。
经液相测纯,纯度达98%以上。
实施例2化合物的化学结构测定
结构测定用JASCO V650型紫外分光光度计测定紫外光谱,在DMSO溶剂中用Brukeravance Ⅲ HD 600HZ型核磁共振仪记录光谱,用AB SCIEX Triple TOF 5600+型高分辨质谱仪测定质谱。
化合物的理化性质无色透明针状晶体(甲醇),薄层板在紫外灯245nm波长照射下有暗斑,在365nm照射下无荧光。UV(CH3OH)λmax=206nm,HR-ESI-MS m/z 375.1817[M+H]+,749.3548[2M+H]+,分子式为C22H22N4O2;1H-NMR(600MHz,DMSO-d6)δ:7.47(1H,d,J=4.2,7a-N),7.15(1H,d,J=7.2,H-4′),7.04(1H,d,J=7.8,H-4),6.92(1H,t,J=7.6,H-6),6.79(1H,t,J=7.6,H-6′),6.70(1H,d,J=4.5,7a′-N),6.60(1H,t,J=7.5,H-5),6.45(1H,t,J=7.1,H-5′),6.41(1H,d,J=8.1,H-7′),6.38(1H,d,J=8.0,H-7),5.48(1H,d,J=3.4,H-8a),4.87(1H,d,J=4.6,H-8a′),3.18(3H,s),2.42(1H,dd,J=11.4,3.7,2′),2.29(3H,s,N1′-CH3),2.09(1H,td,J=11.8,3.2,H-2′),1.98(1H,td,J=12.7,4.9,H-3′),1.59(1H,d,J=12.4,H-3′)。13C-NMR(151MHz,DMSO-d6)δ:158.0(C-2),186.3(C-3),127.0(C-4),118.7(C-5),129.3(C-6),115.5(C-7),51.5(C-3a),142(C-7a),66.3(C-8a),34.6(N1-CH3),45.4(C-2′),33.6(C-3′),126.0(C-4′),115.9(C-5′),127.3(C-6′),112.3(C-7′),34.8(C-3a′),146.0(C-7a′),69.2(C-8a′),42.6(N1′-CH3)。
从HMBC谱图、HSQC谱图、CD谱图数据并结合上述理化数据,确证化合物的结构式如下:
实施例3化合物抗甲型流感病毒H1N1作用的细胞实验
实验材料:病毒株:A型流感病毒H1N1亚型(A/PuertoRico/8/1934)。
细胞模型:狗肾细胞MDCK(ATCC CCL-34)培养条件:DMEM+10%胎牛血清、37℃、5%CO2。
实验主要仪器及试剂:(Promega,底物货号G755B,批号0000174838;稀释液货号G756B,批号0000173700)试剂盒、生物安全柜(哈尔滨东联电子技术开发有限公司BSC-1360-LIIB2)、EnSpire(Perkin-Elmer)多功能酶标仪、MUNANA Sigma-Aldrich、DMEM培养基(Gibco,货号12800-017,批号1791920)、胎牛血清(FBS,BiologicalIndusturies,货号04-001-1ACS,批号1534372)、化合物蜡梅碱-2,3二酮。
实验方法:
MUNANA溶液的配制
MUNANA母液配制:将25mg MUNANA(Sigma货号:M8639)加入25.54mL纯水中溶解配制成2mM的母液,20μl每管分装后,-20℃保存。
MUNANA稀释液:称取634.725mg MES和44.396mg CaCl2溶于100ml蒸馏水中,使各自终浓度分别为33mM和4mM,调pH为5.6备用。
1.样品的细胞毒性检测
实验采用CellTiter-GloTM(Promega)试剂盒检测样品对细胞的毒性作用。
实验原理:CellTiter-Glo试剂盒通过对ATP进行定量测定来检测培养物中活细胞数目。活细胞可通过代谢等途径产生ATP,试剂盒中使用萤光素酶生成的稳定辉光型信号,萤光素酶生成稳定信号需要ATP的共同作用。向细胞培养基中加入CellTiter-Glo试剂测量发光值,光信号和体系中ATP量成正比,而ATP量和活细胞数呈正相关,因此光信号值可以反映活细胞的数目。
实验步骤:将MDCK细胞接种于96孔细胞培养板中,细胞贴壁后备用。药物用DMEM培养基从100mg·mL-1起连续3倍梯度稀释6个梯度。将药物及适量培养基加入到细胞中,于37℃的CO2培养箱中培养。加药培养24h后,显微镜下观察药物引起的细胞病变效应(CPE),加入CellTiter-Glo检测细胞存活率。药物对细胞的毒性以细胞的活性表示。
计算公式:细胞活性(%)=药物组数值/细胞对照组平均值×100。
2.样品的抗病毒活性检测
实验原理:实验采用测定流感病毒蛋白表达水平来检测病毒的复制水平。流感病毒的结构蛋白的表达水平与病毒的复制成正比;实验采用高灵敏度的试剂检测流感病毒蛋白的表达,通过荧光强度的变化反应出来。
方法步骤:MDCK细胞接种于96孔细胞培养板中,37℃培养过夜后备用。MDCK细胞中同时加入药物及H1N1病毒液。置于37℃细胞培养箱培养24h后,取培养液上清进行检测。
实验设空白对照孔(正常细胞),病毒对照孔(病毒感染后未加药物),阳性药物对照孔(感染后加利巴韦林)。
抑制率(%)=100-(样品孔数值-空白对照数值)/(病毒对照数值-空白对照数值)×100。
3.结果
对甲型流感病毒H1N1的抑制作用结果如图7所示(图A为细胞存活率,图B为细胞抑制率),结果表明本发明所述生物碱类化合物蜡梅碱-2,3二酮对甲型流感病毒H1N1具有明显的抑制作用,表现出明显的量效关系。作用于甲型流感病毒H1N1感染的MDCK细胞的EC50和CC50分别为29.92μg/ml、331μg/ml。实验结果表明,本发明的化合物具有抗甲型流感H1N1活性。
Claims (10)
2.如权利要求1所述的化合物,其特征在于还可形成酸式盐。
3.如权利要求2所述的化合物,其特征在于所述药学上可成的盐选自盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硝酸盐、磷酸盐、乙酸盐,苯甲酸盐,马来酸盐,富马酸盐,苹果酸盐,柠檬酸盐,草酸盐,乳酸盐,琥珀酸盐,酒石酸盐,烷基磺酸盐、芳基磺酸盐,氨基酸盐。
4.如权利要求1所述的化合物的制备方法,其特征在于包括如下步骤:
(1)取山蜡梅叶,用水和/或醇提取,得提取物;
(2)将步骤(1)所得提取物进行酸提,过滤得酸水部分;
(3)将步骤(2)所得酸水部分用碱调节pH至7-12,使用三氯甲烷萃取,取三氯甲烷部位;
(4)将步骤(3)所得三氯甲烷部分加硅胶H拌样,装柱,进行硅胶柱色谱分离,依次采用体积比300:1、300:2、300:4、300:8、300:16、300:32、300:64、300:128、300:250、300:500、300:1000的二氯甲烷-乙醇溶液各洗脱1-3个柱体积,后再用甲醇洗脱1-5个柱体积,通过薄层硅胶色谱TCL检测、合并相同的馏分,依次得到7个馏分;
(5)取步骤(4)所得的第2馏分,通过凝胶柱色谱分离,采用甲醇溶液洗脱1-5个柱体积,依次得到两个馏分,取第2馏分,经半制备高效液相色谱C18反相色谱柱,流动相为体积比为40:60的甲醇-水溶液,检测波长230nm,得到权利要求1所述化合物。
5.如权利要求4所述的制备方法,其特征在于步骤(1)采用体积百分比为80%-95%的乙醇水溶液进行提取。
6.如权利要求4所述的制备方法,其特征在于步骤(2)采用质量体积百分比为2%的盐酸进行酸提。
7.如权利要求4所述的制备方法,其特征在于步骤(3)使用浓氨水调节pH。
8.如权利要求4所述的制备方法,其特征在于步骤(3)调节pH至9。
9.如权利要求4所述的制备方法,其特征在于步骤(5)色谱条件为ODS柱,5mm,i.d.20mm×250mm,流速为9mL/min,权利要求1所述化合物的保留时间为37min。
10.如权利要求1-3任一项所述的化合物在制备抗甲型流感病毒H1N1药物中的应用。
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