CN114874213B - 一种苦参生物碱、苦参提取物制备及抗炎、止痒应用 - Google Patents
一种苦参生物碱、苦参提取物制备及抗炎、止痒应用 Download PDFInfo
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Abstract
本发明公开了由干燥苦参茎制备苦参提取物方法、由活性追踪法制备苦参生物碱类化合物的方法、以及苦参碱类化合物的组合物在抗炎、止痒类化妆品、药品中的应用。本发明公开的苦参生物碱及苦参生物碱+苦参提取物组合物在抗炎、止痒方面效果优秀,苦参生物碱与苦参提取物表现出显著的协同作用,在抗炎、止痒方面具有重要意义。本发明公开的苦参生物碱与苦参提取物组合物及其制剂可广泛应用于抗炎、止痒类化妆品或药品。
Description
技术领域
本发明涉及一种苦参生物碱、苦参提取物制备方法和在抗炎、止痒化妆品或药品中的应用,更具体地讲,本发明涉及由干燥苦参茎制备的苦参生物碱、苦参提取物及其在抗炎、止痒化妆品或药品中的应用。
背景技术
皮肤瘙痒症是由多种因素引起的皮肤病理、生理反应,无明显的原发性损害。西医治疗皮肤瘙痒主要用抗组胺药物,虽能治愈,但具有一些副作用。中医药治疗皮肤病的历史悠久,经验丰富,可控制反复发作性皮肤病或延长其复发时间,减轻或避免化学药的副作用,延缓病变进程,延长存活时间。
苦参为豆科植物苦参的干燥根,具有清热、抗菌等功效,苦参提取物和苦参碱常用作杀菌剂等农药。
例如,中国专利202010364546.0公开了一种天然环保的不结球白菜种子包衣剂及其制备方法,包括成膜剂、引发剂、杀虫杀菌剂、警戒色以及水;其中,成膜剂为2%-10%的蜡质玉米淀粉,引发剂为50mg/L或80mg/L的抗坏血酸,杀虫杀菌剂为20mg/L-300mg/L的苦参碱,警戒色为1%-25%的氧化铁黄。苦参碱天然环保,与抗坏血酸相互作用,能有效的提高不结球白菜种子活力。
中国专利201910372474.1公开了一种防治马铃薯晚疫病的杀菌剂及其制备方法,该杀菌剂以苦参碱、亚麻木脂素和迷迭香素为原药有效成分,三者之间呈现协同增效的抑菌和防治作用,不仅对马铃薯晚疫病的离体致病菌具有较好的抑菌活性,且田间防治实验也显示,该杀菌剂能有效的防治马铃薯晚疫病,防治效果安全显著。
中国专利申请201810281834.2公开了一种消毒杀菌剂配方及其制备方法,该申请以具有杀菌消毒作用的金银花、连翘、大葱、鱼腥草、蒲公英、柴胡、板蓝根、生姜、艾叶、桉树叶、苦参、芦荟等价廉易得、消毒杀菌效果明显的原料为主,高锰酸钾、双氧水、山核桃油为辅,并复配有甘油和二甲基甲酰胺,制得的消毒杀菌液。
苦参也是常用的传统中药,含有多种化学成分并具有广泛的生物活性。近年来,人们也在研究和关注苦参碱在其它方面的应用。
例如,中国专利201910433332.1公开了一种抗炎舒敏修复组合物及其制备方法和应用,该组合物由仙人掌提取物、燕麦麸皮提取物、苦参提取物、麦冬根提取物、芍药根提取物、黄芩根提取物、丁二醇、戊二醇和水组成。
中国专利申请202111508063.4公开了一种抗炎舒敏修护组合物,其包括以下活性成分:仙人掌提取物;燕麦麸皮提取物;苦参提取物;麦冬根提取物;芍药根提取物;黄芩根提取物;鸭皂树提取物。
中国专利申请202210107975.9公开了一种抗敏止痒修复植物提取物,由苦参、蛇床子、甘草、洋甘菊、积雪草、黄连、黄柏、花椒、盐肤木、樟树经粉碎、提取、多级膜分离纯化而得,主要活性成分为苦参碱、氧化苦参碱、积雪草苷、甘草酸、α-红没药醇、蛇床子素、小檗碱、没食子酸和右旋龙脑。各活性成分间合理复配、以协同增效。
上述现有技术中的抗炎舒敏组合物是由多种植物提取物组成的,所含的活性成分也较多。然而,成分越多,所带来的副作用也难以预计。准确确定各活性成分特有的生物活性和药理作用,是准确确定化妆品或药品中合理成分的关键。
虽然近年来国内外对苦参作了较多的研究,然而,苦参生物碱类化合物是否具有抗炎、舒缓、止痒的功效未见报道。
因此,有必要对苦参生物碱类化合物进行有区别的分离、富集,以开发具有抗炎、舒缓、止痒的苦参生物碱类化合物或其组合物应用于化妆品或药品具有重要的应用价值。
发明内容
本发明的发明目的之一是提供一种苦参提取物及其制备方法;本发明的另一发明目的是提供分离、富集苦参生物碱类化合物的方法以及这些苦参生物碱类化合物的组合物;本发明的再一发明目的是提供苦参提取物、苦参生物碱类化合物的组合物在制备消炎、止痒类化妆品或药品中的应用。
一方面,为了实现上述的发明目的,本发明提供了一种苦参提取物,该提取物是采用如下过程制备的:
(1)将干燥苦参茎粉碎;
(2)粉碎后的苦参茎用乙醇-水进行渗漉提取;
(3)合并减压浓缩步骤(2)所得的提取液,得到浸膏;
(4)用水溶解步骤(3)所得的浸膏,得到混悬液,用HCl调节 pH至弱酸性;
(5)步骤(4)所得的混悬液中加入氯仿使分层;
(6)将步骤(5)所得的水层用NH3·H2O调pH至弱碱性,再用氯仿萃取,得到总碱浸膏;
(7)将步骤(6)所得的总碱浸膏经硅胶柱色谱分离,用氯仿- 甲醇进行梯度洗脱,获得若干个馏分;
(8)构建2,4-二硝基氯苯诱导的正常人皮肤成纤维细胞(NHDF) 损伤模型,评价步骤(7)所得的各馏分的保护活性;
(9)将步骤(8)中具有最佳保护活性的馏分经大孔树脂分离,进一步用乙醇-水洗脱,进一步得到不同部位的若干个馏分;
(10)基于前述构建的2,4-二硝基氯苯诱导的正常人皮肤成纤维细胞(NHDF)损伤模型,评价步骤(9)所得的各馏分的保护活性;
(11)经步骤(10)评价具有最佳保护活性的部位的馏分为苦参提取物。
在上述的制备过程中,步骤(1)所使用的苦参茎可以是半干的或干燥的,优选为干燥苦参茎。
在上述的制备过程中,步骤(2)中所采用的乙醇-水为25%-35%的乙醇,例如30%的乙醇;步骤(4)中所述的弱酸性为pH=5-6.5,优选为pH=5.7-6.3,例如pH=6;步骤(6)中所述的弱碱性为pH=9-11,优选为pH=9.5-10.5,例如pH=10。
此处,所说的乙醇浓度为体积浓度,例如30%的乙醇是指乙醇与水的体积比为30:70。
另一方面,为了实现上述的发明目的,本发明还提供了一种苦参生物碱类化合物的制备方法,该方法包括:
A、按上述的方法制备苦参提取物;
B、取步骤A中苦参提取物,经反复硅胶色谱柱、氯仿-甲醇梯度洗脱,再经ODS柱色谱及HPLC分离纯化,得到如式1、式2、式3、式4、式5、式6以及式7所示的苦参生物碱类化合物:
例如,作为本发明制备方法的一种具体实施方式,苦参生物碱、苦参提取物经活性追踪方法从苦参中分离得到:
苦参干燥茎,粉碎,用乙醇-水进行渗漉提取,合并减压浓缩提取液,得浸膏;用水溶解浸膏得混悬液,用HCl调pH到6,并加入氯仿使分层;水层用2%NH3·H2O调pH到10,再用氯仿萃取得总碱浸膏;苦参总碱浸膏经硅胶柱色谱分离,用氯仿-甲醇梯度洗脱获得5 个馏分(Fr.1-5);基于构建的DNCB诱导的NHDF细胞损伤模型,评价 Fr.1-5的保护活性;将具有最佳保护活性的Fr.3经D101大孔树脂分离,用乙醇-水梯度洗脱,得5个馏分;基于构建的DNCB诱导的 NHDF细胞损伤模型,评价5个馏分保护活性;将具有最佳保护活性的50%部位经反复硅胶色谱柱,氯仿-甲醇梯度洗脱,再经ODS柱色谱及HPLC分离纯化,得到结构式如式1、式2、式3、式4、式5、式6、式7所示化合物。
其中,苦参干燥茎粉末与渗漏提取所用酒精的质量比为 1:(10-100);
用于渗漉提取苦参干燥茎粉末所用酒精为25%-35%(酒精:水 -V:V),优选所用酒精为30%(酒精:水-V:V);
苦参总碱提取过程中,用水溶解浸膏得混悬液,用HCl调pH到 5.5-6.5,优选用HCl调pH到6,并加入氯仿使分层,水层用2%NH3·H2O 调pH到9-11,优选调pH到10;
苦参总碱浸膏经硅胶柱色谱分离,选用硅胶为80-100目或 200-300目或300-400目,优选选用硅胶为80-100目;
苦参总碱浸膏经硅胶(80-100目)柱色谱分离,用氯仿-甲醇 (100∶0→0∶100)梯度洗脱,优选用氯仿-甲醇(100∶30→30∶100) 梯度洗脱,更优选用氯仿-甲醇(100∶50→50∶100)梯度洗脱;
保护活性最好的Fr.3经D101大孔树脂分离,用乙醇-水(100∶0 →0∶100)梯度洗脱,优选用乙醇-水(30∶70→50∶50)梯度洗脱,更优选用乙醇-水(50∶50)洗脱;
将具有最佳保护活性的50%部位经反复硅胶色谱柱,氯仿-甲醇梯度洗脱,再经ODS柱色谱及HPLC分离纯化,得到式1、式2、式3、式4、式5、式6、式7所示的化合物。
再一方面,为了实现上述的发明目的,本发明还提供了一种苦参生物碱类化合物的组合物,该组合物包括根据前述方法制备的如式1、式2、式3、式4、式5、式6以及式7所示的化合物;在该组合物中,式1、式2、式3、式4、式5、式6、式7所示的化合物的质量比为 1:1-100:1-100:1-100:1-100:1-100:1-100。
进一步地,在本发明的苦参生物碱类化合物的组合物中,可以进一步含有前述的苦参提取物,该组合物中,式1、式2、式3、式4、式5、式6、式7所示的化合物与苦参提取物的质量比为 1:1-100:1-100:1-100:1-100:1-100:1-100:1-500,优选的质量比为1:1-50:1-50:1-50:1-50:1-50:1-50:1-250,更优选的质量比为1:1-3:1-3:1-3:1-3:1-3:1-3:80-120。
又一方面,为了实现上述的发明目的,本发明还提供了前述的苦参提取物或前述的苦参生物碱类化合物的组合物在制备抗炎、止痒类化妆品或药品中的应用。
本发明的苦参生物碱、苦参提取物能够抑制2,4-二硝基氯苯 (DCNB)诱导的正常人皮肤成纤维细胞(NHDF细胞)炎性因子过度分泌及炎性损伤,发挥抗炎、保护活性。
本发明的苦参生物碱、苦参提取物能够抑制皮肤瘙痒模型小鼠炎性因子分泌,发挥抗炎活性,具有优异的抑制皮肤瘙痒作用。因此可应用于制备抗炎、抗瘙痒化妆品或药物。
在本发明上述的应用中,该应用是通过提高NHDF细胞活力、抑制NHDF细胞炎性细胞因子分泌、抑制NHDF细胞内ROS含量、和/或抑制NHDF细胞NF-κB/MAPKs信号通路激活而实现的。
在本发明上述的应用中,所采用的抗炎、止痒类化妆品或药品可以为外用制剂,其形式可以为膏剂、乳剂、慕斯、喷剂、或液体制剂。在本发明中,苦参生物碱与苦参提取物组合物可以作为化妆品或护肤品添加剂,加入至化妆品或护肤品中,作为抗炎、止痒的有效成分。
在本发明上述的应用中,止痒是指抑制或缓解由皮炎、湿疹、荨麻疹、皮肤干燥症、或浅部真菌感染等皮肤病所引起的痒症。
在本发明上述的应用中,所采用的外用制剂可以为相应的缓控释剂,其形式可以为用于涂抹给药的包合物、脂质体、微球、纳米粒、或乳剂等。
在本发明上述的应用中,止痒药品可以为复方制剂,其包括辅料以及前述的苦参提取物或前述的苦参生物碱类化合物的组合物。
本发明相对于现有技术,具有如下的优点及有益效果:
(1)本发明的苦参生物碱、苦参提取物、苦参生物碱与苦参提取物的组合物能显著抑制皮肤瘙痒小鼠炎性细胞因子的过度分泌;
(2)本发明的苦参生物碱、苦参提取物、苦参生物碱与苦参提取物的组合物能显著减少皮肤瘙痒模型小鼠搔抓次数;
(3)本发明的苦参生物碱与苦参提取物的组合物抗炎止痒活性强于苦参生物碱、苦参提取物单独使用。
下面将结合具体实施方式和附图对本发明技术方案进行清楚、完整描述。本领域技术人员可以理解的是,此处所描述的具体实施方式仅仅是本发明一部分的实施方式,而不是全部的实施方式。基于本发明的精神,本领域的普通技术人员可以在不作出创造性劳动的前提下进行相应的替换、变换、改变或改进,但这些替换、变换、改变或改进,仍属于本发明的保护范围。
附图说明
图1-图8分别是苦参生物碱-1的HR-ESI-MS谱图、1HNMR谱图、13CNMR谱图、DEPT谱图、1H-1H COSY谱、HSQC谱、HMBC谱、NOESY谱;
图9-图16分别是苦参生物碱-2的HR-ESI-MS谱图、1HNMR谱图、13CNMR谱图、DEPT谱图、1H-1H COSY谱、HSQC谱、HMBC谱、NOESY谱;
图17-图24分别是苦参生物碱-3的HR-ESI-MS谱图、1HNMR谱图、13CNMR谱图、DEPT谱图、1H-1H COSY谱、HSQC谱、HMBC谱、NOESY谱;
图25-图32分别是苦参生物碱-4的HR-ESI-MS谱图、1HNMR谱图、13CNMR谱图、DEPT谱图、1H-1H COSY谱、HSQC谱、HMBC谱、NOESY谱;
图32-图40分别是苦参生物碱-5的HR-ESI-MS谱图、1HNMR谱图、13CNMR谱图、DEPT谱图、1H-1H COSY谱、HSQC谱、HMBC谱、NOESY谱;
图41-图48分别是苦参生物碱-6的HR-ESI-MS谱图、1HNMR谱图、13CNMR谱图、DEPT谱图、1H-1H COSY谱、HSQC谱、HMBC谱、NOESY谱;
图49-图56分别是苦参生物碱-7的HR-ESI-MS谱图、1HNMR谱图、13CNMR谱图、DEPT谱图、1H-1H COSY谱、HSQC谱、HMBC谱、NOESY谱;
图57显示了苦参生物碱组合物+苦参提取物的组合物抑制DCNB 诱导的NHDF细胞活力显著降低;
图58显示了苦参生物碱组合物+苦参提取物的组合物抑制DCNB 诱导的NHDF细胞内ROS含量升高;
图59显示了苦参生物碱组合物+苦参提取物的组合物抑制DCNB 诱导的NHDF细胞炎性细胞因子含量升高;
图60显示了苦参生物碱组合物+苦参提取物的组合物抑制DCNB 诱导的NHDF细胞NF-κB/MAPKs信号通路激活;
图61显示了苦参生物碱组合物+苦参提取物的组合物调控DCNB 诱导的小鼠血清IgE及炎性因子含量变化。
具体实施方式
本发明具体实施方式中所采用的主要试验仪器和主要材料来源为:化合物的核磁检测用Bruker AV-500或Bruker AV-400MHz核磁共振仪测定核磁(NMR),并以TMS为内标(德国Bruker公司);用Agilent 6210LC/MSD TOF型质谱仪测定高分辨质谱;用ThermoFinnigan LCQ Advantage MAX质谱仪(美国Thermo公司)测定质谱(ESI-MS);HPLC 采用Dionex型高效液相色谱仪(美国Dionex公司);PHPLC采用 Varian制备型高效液相色谱仪(美国Varian公司)和Agilent 1100LC/MSD型高校色谱仪(美国Agilent公司);Cosmosil C-1(8250mm×4.6mm,5μm)色谱柱。Sephadex LH-20层析填料购于Pharmacia 公司;ODS柱色谱材料购于德国Merck公司;柱色谱用硅胶购于青岛海洋化工厂;核磁用氘代试剂购自美国CIL公司;所用试剂均为分析纯和色谱纯。
实施例1苦参活性生物碱的分离、制备
下面所进行的实施方式是一具体的实施例,但分离制备的方法并不限于该实施例。
苦参干燥茎10kg,粉碎,乙醇(100L,30%)的渗漏提取,合并减压浓缩提取液,得浸膏552g。用水溶解浸膏得混悬液,用HCl 调pH到6,并加入氯仿使分层。水层用2%NH3·H2O调pH到10,再用氯仿萃取得总碱浸膏221g。
苦参总碱200g浸膏经硅胶(1.5kg 800-100目)柱色谱分离,用氯仿-甲醇(100∶50→50∶100)梯度洗脱获得5个馏分(Fr.1-5)。
基于构建的DNCB诱导的NHDF细胞损伤模型,评价Fr.1-5保护活性。
将具有最佳保护活性的Fr.3经D101大孔树脂分离,用乙醇-水 (50∶50)洗脱,得馏分。
实验最佳保护活性的50%乙醇洗脱组分标记为苦参提取物。
实施例2苦参生物碱的分离、制备
下面所进行的实施方式是一具体的实施例,但分离制备的方法并不限于该实施例。
将实施例1获得的具有最佳保护活性的50%乙醇洗脱组分经反复硅胶色谱柱,氯仿-甲醇梯度洗脱,再经ODS柱色谱及HPLC分离纯化,得到式1所示化合物(1.21g)、式2所示化合物(1.01g)、式3所示化合物(989mg)、式4所示化合物(884mg)、式5所示化合物(921mg)、式6所示化合物(753mg)、式7所示化合物(669mg)。
苦参生物碱-1的结构鉴定:苦参生物碱-1为淡黄色油状物,碘化铋钾显色显阳性。 (c 1.00,CH3OH)。HR-ESI-MS显示准分子离子峰m/z 261.1595 [M+H]+(计算值为C15H21N2O2,261.1598),确定分子式为C15H20N2O2,不饱和度为7。
1H NMR(400MHz,CD3OD)谱中显示该化合物除活泼氢外含有19个质子,其中有1个烯烃质子信号在δH7.63(1H,s)处,2个连杂原子的亚甲基氢信号在δH 3.45(4H,m)处。13C NMR(100MHz,CD3OD)谱中共出现15个碳信号,结合DEPT-135谱可知其分别为5个季碳、1 个次甲基和9个亚甲基。其中,包括有5个芳香碳信号(δC 153.8, 150.1,135.7,116.9,115.4),1个羧基碳信号(δC 181.4),2个连杂原子的亚甲基信号(δC 51.4,50.7),及7个亚甲基碳信号(δC 38.1, 31.4,26.5,25.2,23.3,21.0,20.9)。除去5个芳香碳和1个羧基,该化合物的不饱和度还有3,推测该化合物的母核有3个环。
在HMBC谱中,可观察到H-17(δH 7.65)与C-6(δC 153.7)、C-11 (δC 150.1)、C-5(δC116.9)、C-4(δC 25.2)有相关,H-13(δH 1.87) 分别与C-11(δC 150.1)和C-15(δC 181.9)有相关,H-14(δH 2.23) 与C-12(δC 31.4)、C-15(δC 181.9)有相关,H-3(δH 1.98)与C-5 (δC116.9)有相关,H-8(δH 2.75)与C-10(δC 50.7)、C-7(δC 115.4) 有相关,说明双键在C环,且羧基在C-15位。在1H-1H COSY谱中, H-13(δH 1.87)与H-14(δH 2.23)有相关,H-9(δH 1.98)与H-8(δH 2.75)和H-10(δH 3.45)有相关,H-3(δH 1.98)和H-4(δH 2.71) 有相关,进一步验证了化合物结构,命名为苦参生物碱-1。
结合1D和2D NMR谱信息,对苦参生物碱-1的碳氢信号进行了全归属(见下表1):
表1苦参生物碱-1NMR数据(in CD3OD,400MHz,δin ppm,J in Hz)
苦参生物碱-2的结构鉴定:
苦参生物碱-2为黄色油状物,碘化铋钾显色阳性。(c 1.37,CH3OH)。HR-ESI-MS显示准分子离子峰m/z 265.1911[M+H]+(计算值为C15H25N2O2,265.1911),确定分子式为C15H24N2O2,不饱和度为5。
1H NMR(400MHz,CD3OD)谱中显示该化合物含有24个质子信号,包括无叶豆碱型生物碱C-10位特征的氢信号[δH 3.95(1H,dd,J= 13.6,8.0Hz),3.19(1H,m)]。13C NMR(100MHz,CD3OD)谱中共出现15个碳信号,结合DEPT-135谱可知为1个季碳、4个次甲基和 10个亚甲基。其中包括1个酰羰基碳信号(δC 172.6),2个连杂原子的次甲基碳信号(δC 60.6,74.0),以及3个连杂原子的亚甲基碳信号(δC 48.2,59.5,72.2)。除去1个酰羰基后,该化合物的不饱和度还有4,推测该化合物的母核有4个环。
在HMBC谱中,可观察到H-10a(δH 3.95)与C-6(δC 60.6)、 C-8(δC 31.0)有相关,H-11(δH 2.87)分别与C-7(δC 36.8)、C-10 (δC 48.2)、C-13(δC 20.1)、C-14(δC 24.7)有相关,H-3(δH 2.34) 与C-6(δC 60.6)、C-10(δC48.2)有相关,证实其结构。在1H-1H COSY 谱中,H-7(δH 2.74)与H-6(δH 3.21)和H-8b(δH 1.36)有相关, H-9(δH 2.35)与H-11(δH 2.87)和H-10a(δH 3.95)有相关,进一步验证其结构。NOESY谱中,可以看到H-9(δH 2.35)与H-11(δH2.87) 和H-7(δH 2.74)有相关,但H-9(δH 2.35)与H-6(δH 3.21)没有相关,推测H-9、H-11、H-7处于同侧。最终鉴定化合物的结构,命名为苦参生物碱-2。
结合1D和2D NMR谱信息,对苦参生物碱-2的碳氢信号进行了全归属(见下表2):
表2苦参生物碱-2NMR数据(in CD3OD,400MHz for 1H,δin ppm,J in Hz)
苦参生物碱-3的结构鉴定:
苦参生物碱-3为黄色油状物,碘化铋钾显色显阳性。(c 1.05,CH3OH)。HR-ESI-MS显示准分子离子峰m/z 265.1909[M+H]+(计算值为C15H25N2O2,265.1911),确定分子式为 C15H24N2O2,不饱和度为5。
1H NMR(400MHz,CD3OD)谱中显示,该化合物含有24个质子信号,其中包括一个连氧次甲基氢信号[δH 3.97(1H,dd,J=11.2,5.6 Hz)]。此外,1H NMR谱还显示有生物碱C-10位特征的氢信号[δH 3.43 (1H,dd,J=13.6,10.8Hz),3.28(1H,m)]。13C NMR(100MHz, CD3OD)谱中共出现15个碳信号,结合DEPT-135谱可知其为1个季碳、5个次甲基和9个亚甲基。其中,包括有1个酰羰基碳信号(δC 173.2),3个连杂原子的次甲基信号(δC 68.9,62.7,58.1),以及连3个杂原子的亚甲基碳信号(δC 56.5,50.0,48.7)。除去1个酰羰基(δC 173.2)和1个连氧的次甲基(δC 68.9),该化合物的不饱和度还有4,推测该化合物的母核有4个环。
在HMBC谱中,可观察到H-10a(δH 3.43)与C-8(δC 32.1)、C-11 (δC 62.7)有相关,H-5(δH 3.97)与C-2(δC 173.2)、C-6(δC 58.1)、 C-8(δC 32.1)、C-9(δC 29.3)有相关,证实羟基位于C-5位上,羰基位于C-2位上。在1H-1H COSY谱中,可以看到H-5(δH 3.97)与H-4 (δH1.98)有相关,H-9a(δH 1.90)与H-11(δH 2.22)有相关,进一步证实了其结构。NOESY谱中,可以看到H-3a(δH 1.61)与H-5(δH 3.97)和H-6(δH 3.33)有相关,推测5-OH是α取向。至此,可以确定化合物的结构,命名为苦参生物碱-3。
结合1D和2D NMR谱信息,对苦参生物碱-3的碳氢信号进行了全归属(见下表3):
表3苦参生物碱-3NMR数据(in CD3OD,400MHz,δin ppm,J in Hz)
苦参生物碱-4的结构鉴定:
苦参生物碱-4为黄色油状物,碘化铋钾显色显阳性。 (c1.05,CH3OH)。HR-ESI-MS显示准分子离子峰m/z 265.1914 [M+H]+(计算值为C15H25N2O2,265.1911),确定分子式为C15H24N2O2,不饱和度为5。
1H NMR(400MHz,CD3OD)谱中显示出23个质子信号,包括一个连氧的次甲基氢信号[δH 3.97(1H,dd,J=10.4,5.2Hz)]。另外,生物碱C-10位特征的氢信号[δH 3.47(1H,m),3.08(1H,t,J= 11.6Hz)]。13C NMR(100MHz,CD3OD)谱中共出现15个碳信号,结合DEPT-135谱可知其分别为1个季碳、5个次甲基和9个亚甲基。其中包括1个酰羰基碳信号(δC 173.6),3个连杂原子的次甲基碳信号(δC 69.2,64.5,57.6),以及3个连杂原子的亚甲基碳信号(δC57.0,51.3,49.1)。除去1个酰羰基(δC 173.6)和1个连氧的次甲基(δC 69.2),该化合物的不饱和度还有4,推测该化合物的母核有4个环。
在HMBC谱中中,可观察到H-3(δH 3.97)与C-4(δC 29.1)、C-2 (δC 173.6)有相关,H-10b(δH 3.08)与C-9(δC 31.8)、C-11(δC 64.5) 有相关,H-5b(δH 1.45)与C-3(δC 69.2)、C-6(δC 57.6)、C-7(δC 42.9)、C-4(δC 29.1)有相关,证实其结构。在1H-1H COSY谱中中,可观察到H-4b(δH1.65)与H-3(δH 3.97)和H-5a(δH 1.56)有相关, H-7(δH 1.81)与H-6(δH 3.44)和H-8b(δH 1.07)有相关,进一步证实了化合物的结构。NOESY谱中,可以看到H-3(δH 3.97)与H-5b (δH 1.45)和H-6(δH 3.44)有相关,H-7(δH 1.81)与H-5a(δH 1.56) 有相关,推测3-OH为β构型,于是得出化合物的相对构型。至此,可确定化合物的结构,命名为苦参生物碱-4。
结合1D和2D NMR谱信息,对苦参生物碱-4的碳氢信号进行了全归属(见下表4):
表4苦参生物碱-4NMR数据(in CD3OD,400MHz,δin ppm,J in Hz)
苦参生物碱-5的结构鉴定:
苦参生物碱-5为淡黄色油状物,碘化铋钾显色显阳性。 (c1.05,CH3OH)。HR-ESI-MS显示准分子离子峰m/z 209.1654 [M+H]+(计算值为C12H21N2O,209.1648),确定分子式为C12H20N2O,不饱和度为4。
在1H NMR(400MHz,CD3OD)谱显示共有20个氢质子,其中包含一个甲基氢信号[δH2.09(3H,s)],及C-10位特征氢信号[δH 4.53 (1H,d,J=15.6Hz),2.87(1H,m)]。在13C NMR(100MHz,CD3OD) 谱中显示共有20个碳原子,结合DEPT谱,可知包括1个季碳,1 个甲基,7个次甲基和3个亚甲基。其中,包括1个羰基碳信号(δC 172.3),1个与杂原子相连的次甲基碳信号(δC 60.6),3个与杂原子相连的亚甲基碳信号(δC 62.4,60.6,57.5),及1个甲基碳信号(δC 47.5)。除去1个羰基(δC 172.3)后,该化合物的不饱和度还有3,推测该化合物的母核3个环。
在HMBC谱中,观察到H-14(δH 2.09)与C-11(δC 62.4)、C-13 (δC 57.5)有相关,H-13(δH 3.11)与C-11(δC 62.4)、C-8(δC 33.6) 有相关,H-6(δH 3.56)与C-5(δC 28.9)、C-13(δC 57.5)有相关, H-10a(δH 4.53)与C-2(δC 172.3)、C-6(δC 60.6)、C-8(δC 33.6)、 C-9(δC 30.8)、C-11(δC 62.4)有相关,证实其结构。在1H-1H COSY 谱中,观察到H-5(δH 1.84)与H-6(δH 3.56)有相关,H-9(δH 1.96) 与H-10a(δH 4.53)、H-11(δH 2.89)有相关,H-3a(δH2.33)与H-4a (δH 1.87)、H-4b(δH 1.69)有相关,进一步证实其结构。在NOESY 谱中中,H-6(δH 3.56)与H-9(δH 1.96)没有相关,可以判断化合物的相对构型。至此,确定化合物示的结构,命名为苦参生物碱-5。
结合1D和2D NMR谱信息,对苦参生物碱-5的碳氢信号进行了全归属(见下表5):
表5苦参生物碱-5NMR数据(in CD3OD,400MHz,δin ppm,J in Hz)
苦参生物碱-6的结构鉴定:
苦参生物碱-6为黄色油状物,碘化铋钾显色阳性反应。 (c 1.05,CH3OH)。HR-ESI-MS显示准分子离子峰m/z 263.1753 [M+H]+(计算值为C15H23N2O2,163.1754),确定分子式为C15H22N2O2,不饱和度为6。
1H NMR(400MHz,CD3OD)谱中显示该化合物除活泼氢外有21个质子,包括1个烯烃质子信号[δH 7.42(1H,s)],2个与杂原子相连的亚甲基氢信号[δH3.56(2H,m),3.47(2H,m)]。13CNMR(100MHz, CD3OD)谱中共出现15个碳信号,结合DEPT-135谱可知其分别为3个季碳、3个次甲基和9个亚甲基。其中,包括1个羧基碳信号(δC181.9),3个烯碳信号(δC 165.0,155.3,97.8),1个与杂原子相连的次甲基碳信号(δC 57.1),以及2个与杂原子相连的亚甲基碳信号(δC 53.1,52.4)。
与苦参生物碱-1比较,发现少了2个季碳,多出了2个次甲基,推测苦参生物碱-1环上有1个双键变成了单键形成了苦参生物碱-6。在HMBC谱中,H-17(δH 7.42)与C-4(δC23.0)、C-5(δC 97.8)、 C-6(δC 165.0)和C-11(δC 57.1)有相关,H-4b(δH 2.05)与C-6(δC165.0)和C-10(δC 52.4)有相关,证明了C-7与C-11间是单键。在1H-1H COSY谱中,H-7(δH2.71)与H-11(δH 3.39)有相关,H-13 (δH 1.84)与H-14(δH 2.21)有相关,H-3(δH 1.97)和H-4(δH 2.77) 有相关,进一步验证其结构。在NOESY谱中,H-7(δH 2.70)与H-11 (δH 3.39)没有相关,与H-12(δH 2.38)有相关,故H-7与H-11不在同一侧。至此确定化合物的结构,命名为苦参生物碱-6。
结合1D和2DNMR谱信息,对苦参生物碱-6的碳氢信号进行了全归属(见下表6):
表6苦参生物碱-6NMR数据(in CD3OD,400MHz,δin ppm,J in Hz)
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苦参生物碱-7的结构鉴定:
苦参生物碱-7为黄色油状物,碘化铋钾显色显阳性。(c 1.00,CH3OH)。HR-ESI-MS显示准分子离子峰m/z 263.1756[M+H]+(计算值为C15H23N2O2,263.1754),确定分子式为C15H22N2O,不饱和度为6。
1H NMR(400MHz,CD3OD)谱中显示,该化合物除活泼氢外含有 21个质子,其中包括苦参碱型生物碱C-17位的特征氢信号[δH3.51(1H,m)、3.09(1H,m)],及2个与杂原子相连的亚甲基氢信号 [δH 3.45(4H,m)]。13C NMR(100MHz,CD3OD)谱中共出现15个碳信号,结合DEPT-135谱可知其分别为4个季碳、1个次甲基和10个亚甲基。其中包括1个羧基碳信号(δC181.3),3个烯碳信号(δC169.9,163.8,96.4),以及3个与杂原子相连的亚甲基碳信号(δC52.6,52.3,45.4)。与苦参生物碱-1比较,大部分碳信号相吻合,发现只是少了1个季碳C-5(δC 115.4),多出了1个亚甲基C-13(δC 45.4),推测苦参生物碱-7中C-5与C-13间的双键被还原成单键。
在HMBC谱中,可观察到H-17b(δH 3.07)与C-5(δC 35.7)、C-11 (δC 163.8)、C-4(δC23.5)有相关,H-17a(δH 3.51)与C-5(δC35.7)、C-11(δC 163.8)、C-6(δC 169.9)有相关,证实了与苦参生物碱-1比较,苦参生物碱-7的C环上C-5与C-13间是单键。在1H-1H COSY谱中,H-17b(δH 3.07)与H-5(δH 2.79)有相关,H-13(δH 1.81) 与H-14(δH 2.20)有相关,H-3(δH1.35)和H-4(δH 1.91)有相关,进一步验证了上述结构。至此,确定化合物结构,命名为苦参生物碱 -7。
结合1D和2D NMR谱信息,对苦参生物碱-7的碳氢信号进行了全归属(见下表7):
表7苦参生物碱-7NMR数据(inCD3OD,400MHz,δin ppm,J in Hz)
实验例3:苦参生物碱、苦参提取物、
苦参生物碱+苦参提取物的组合物的抗炎活性的细胞试验
5×103细胞/孔,NHDF细胞(正常人皮肤成纤维细胞)接种于 96孔细胞培养板,培养24h。加入2,4-二硝基氯苯(10μM)处理细胞8h。收集细胞,冷PBS清洗2遍,加入新的培养基并加入终浓度为10μg/mL的苦参生物碱组合物(苦参生物碱-1:苦参生物碱-2:苦参生物碱-3:苦参生物碱-4:苦参生物碱-5:苦参生物碱-6:苦参生物碱-7质量比为1:1:1:1:1:1:1)或苦参提取物或苦参生物碱组合物+苦参提取物的组合物(苦参生物碱组合物与苦参提取物质量比为 1:100)共培育24h后,分离细胞及上清液备用。
本实验部分,苦参生物碱组合物组标记为KSJ;空白组标记为 CON,2,4-二硝基氯苯组标记为DCNB;苦参提取物组标记为KST;苦参生物碱组合物与苦参提取物的组合物组标记为KSJT。
图57的实验结果显示,相比于空白对照组(细胞活力设为100%), DCNB暴露使NHDF细胞活力显著降低(22.3%),而KSJ,KST,KSJT 组细胞活力高于DCNB组;其中KSJT组细胞活力提高最为显著,高于同浓度的KSJ,KST组;相比于DCNB组,KSJT组细胞活力提高17.5%。
ROS是诱导细胞氧化损伤、细胞凋亡的毒性因子。图58的实验结果显示,相比于CON组(设为100%),DCNB暴露使NHDF细胞内 ROS含量显著增加(383%),而相比于DNCB组,KSJ,KST,KSJT组细胞内ROS含量显著降低;其中KSJT组细胞内ROS含量显著减少;相比于DCNB组,KSJT组细胞内ROS含量减少54.2%。
白细胞介素在免疫调节和炎症应答中具有重要作用,细胞因子分为前炎性细胞因子和抗炎性细胞因子两类,是机体正常防御系统的重要组成部分。前炎性细胞因子主要包括IL-1、IL-5、IL-6、IL-8、 IFN-γ、TNF-α等,具有促进炎症反应的活性。抗炎性细胞因子主要包括IL-4、IL-10、IL-13等,具有抑制炎症反应活性。
图59的实验结果显示,DNCB暴露使NHDF细胞炎性细胞因子 (IL-5、IL-6、IFN-γ、TNF-α、IL-4及IL-13)及RANTES、TARC 分泌显著增加,实验结果说明DCNB暴露诱导NHDF细胞炎性损伤。而KSJ,KST,KSJT处理后,细胞炎性因子及RANTES、TARC含量升高被抑制。其中花KSJT对DNCB诱导的炎性因子过度分泌及RANTES、TARC 含量升高抑制活性最显著,且高于KSJ,KST单独使用。我们推测,苦参生物碱、苦参提取物可能是通过发挥协同作用发挥抑制炎性因子过度分泌及RANTES、TARC含量升高的。
NF-κB则信号通路是介导炎症反应关键的转录因子,激活的NF- κB信号通路会进一步上调炎性因子的转录翻译,促进炎症。大量研究显示NF-κB、信号转导子和转录激活因子-1(STAT-1)以及丝裂原活化蛋白激酶(MAPK)信号通路相关蛋白的表达在某些药物/活性天然分子发挥抗炎过程中有着重要作用。与丝裂原活化蛋白激酶 (MAPK)一样,STAT-1在皮肤炎症中细胞因子和趋化因子的产生中发挥重要作用。因此,通过抑制促炎细胞因子和趋化因子的产生,通过转录因子的转录调节,如调节NF-kB、STAT-1和MAPKs通路中蛋白的表达,是治疗炎症性皮肤病的有效方法。
图60的实验结果显示,DCNB暴露使NHDF细胞NF-κB信号通路中IκB-α蛋白的含量显著减少,说明DCNB暴露后诱导NHDF细胞NF- κB信号通路激活。而KSJ,KST,KSJT处理后,NHDF细胞NF-κB信号通路中IκB-α蛋白减少被抑制,说明DCNB暴露后诱导的NHDF细胞NF-κB信号通路激活被抑制。
进一步的,DCNB暴露诱导NHDF细胞STAT-1蛋白磷酸化,总 STAT-1蛋白含量显著减少,说明DCNB暴露对NHDF细胞STAT-1蛋白有着重要的影响。而KSJ,KST,KSJT处理后,NHDF细胞中STAT-1 总蛋白减少被抑制,STAT-1蛋白磷酸化被抑制,说明DCNB暴露后诱导的NHDF细胞STAT-1蛋白表达失调被抑制。
同时,我们检测了MAPK信号通路相关蛋白表达变化,实验结果显示,DCNB暴露显著诱导NHDF细胞MAPK信号通路p38,JNK,ERK 蛋白磷酸化,总p38,总JNK,总ERK蛋白含量显著减少,说明DCNB 暴露对NHDF细胞MAPK信号通路蛋白表达有重要的影响。而KSJ,KST, KSJT处理后,NHDF细胞中MAPK信号通路总p38,JNK,ERK蛋白减少被抑制,p38,JNK,ERK蛋白磷酸化显著缓解,说明DCNB暴露后诱导的NHDF细胞MAPK信号通路蛋白表达失调被有效调节。
我们推测,KSJ,KST,KSJT对DNCB诱导的NHDF细胞NF-κB信号通路激活具有有效的抑制作用,且KSJ,KST,KSJT对DNCB诱导的 NHDF细胞MAPK及信号通路相关蛋白及STAT-1蛋白的表达失控也具有有效的调节活性,KSJ,KST,KSJT对DNCB诱导的NHDF细胞炎性因子分泌失调可能是通过调控NF-κB,MAPK信号通路及STAT-1蛋白的表达实现的。
实验例4苦参生物碱、苦参提取物、
苦参生物碱+苦参提取物的组合物的抗炎、止痒活性的动物试验
昆明种小鼠,SPF级,雄性,体重(21±2)g,恒温环境饲养,温度(20±2)℃,湿度40%-70%,饲养环境为光照节律12h:12h(7: 00-19:00),自由进食进水,适应性喂养1周。将小鼠随机分为5组,每组8只,分别为正常组、皮肤瘙痒模型组、苦参生物碱组合物组(苦参生物碱-1:苦参生物碱-2:苦参生物碱-3:苦参生物碱-4:苦参生物碱-5:苦参生物碱-6:苦参生物碱-7质量比为1:1:1:1:1:1:1)、苦参提取物组、苦参生物碱组合物与苦参提取物组合物(苦参生物碱组合物与苦参提取物质量比为1:100)组。实验开始后,除正常组外,剃须后,用200μL 2%DNCB溶液在1×1cm贴片上涂抹小鼠背部皮肤1周,并用200μL 0.2%DNCB溶液每周两次再次激发。
第21天开始,各用药组小鼠按0.2mg/cm2涂抹给药,于每日傍晚给药1次,连续14天(正常组、皮肤瘙痒模型组每日给予同等剂量蒸馏水)。实验过程中,小鼠颈背部剃毛,保持颈背部无毛。于第 28天涂药30min后,记录小鼠搔抓表现;摘眼球取血,3000r/min 离心10min,取血清,ELISA法检测血清IgE、IL-6、IL-5及IL-13 含量。
本实验部分,苦参生物碱组合物标记为KSJ;空白组标记为CON, 2,4-二硝基氯苯标记为DCNB;苦参提取物标记为KST;苦参生物碱组合物与苦参提取物组合物标记为KSJT。
从表8数据中可以看出,与正常组比较,皮肤瘙痒模型组瘙痒潜伏时间显著缩短,瘙痒次数明显增多。而KSJT,KSJ,KST用药后小鼠皮肤瘙痒潜伏时间显著延长,小鼠皮肤瘙痒次数明显减少,其中 KSJT组小鼠皮肤瘙痒潜伏时间延长最长,小鼠皮肤瘙痒次数减少最为显著。
表8苦参生物碱组合物+苦参提取物的组合物对小鼠瘙痒潜伏时间及瘙痒次数的影响
注:与CON组比较#p<0.05,##p<0.01;与DCNB组比较*p<0.05,**p<0.01.
大量研究发现,模型小鼠在皮炎发作后均会表现出血清中IgE水平升高的现象。因此,我们检测KSJT,KSJ,KST用药能否调控DCNB 介导的皮炎小鼠血清IgE水平及相关炎性因子IL-6、IL-5及IL-13 含量变化。
从图61的分析实验结果可以发现,相比于正常组,皮肤瘙痒模型组血清IgE含量显著性升高。而KSJT,KSJ,KST处理后,与DCNB 组比较,小鼠血清IgE含量显著降低。其中,KSJT组IgE含量变化最为显著。我们推测,在相同浓度条件下,KSJ与KST可发挥协同抗炎活性。
从图61数据中可以看出,与正常组比较,皮肤瘙痒模型组小鼠血清炎性细胞因子IL-6、IL-5及IL-13含量显著性升高,说明DCNB 模型组小鼠受到严重的炎性损伤。而与皮肤瘙痒模型DCNB组比较, KSJT,KSJ,KST用药后炎性细胞因子IL-6、IL-5及IL-13含量升高被显著抑制,说明KSJT,KSJ,KST均具有抑制DCNB诱导的小鼠皮肤炎性损伤的活性。此外,KSJT抑制小鼠皮肤瘙痒模型小鼠炎性因子 IL-6、IL-5及IL-13分泌活性高于KSJ,KST。
皮肤瘙痒症发生与免疫细胞有重要的关系,肥大细胞广泛存在于皮肤和组织血管周围,内含组胺等炎性因子。PAR-2是G蛋白偶联受体家族成员,PAR-2可促进血管内皮白细胞黏附迁移,促进IL-1等表达与释放,具有重要的促炎效应和介导组织损伤的作用。
从表9数据中可以看出,与正常组比较,DCNB组PAR-2含量显著性升高。KSJT,KSJ,KST用药后,与皮肤瘙痒模型DCNB组比较,KSJT, KSJ,KST组PAR-2含量显著降低。相比于KSJ,KST组,KSJT组PAR-2 降低最显著。
表9苦参生物碱组合物+苦参提取物的组合物对小鼠血清PAR-2的影响
注:与CON组比较#p<0.05,##p<0.01;与DCNB组比较*p<0.05,**p<0.01。
Claims (2)
1.一种苦参生物碱类化合物的制备方法,该方法包括:
(1)将干燥苦参茎粉碎;
(2)粉碎后的苦参茎用乙醇-水进行渗漉提取;
(3)合并减压浓缩步骤(2)所得的提取液,得到浸膏;
(4)用水溶解步骤(3)所得的浸膏,得到混悬液,用HCl调节pH至弱酸性;
(5)步骤(4)所得的混悬液中加入氯仿使分层;
(6)将步骤(5)所得的水层用NH3·H2O调pH至弱碱性,再用氯仿萃取,得到总碱浸膏;
(7)将步骤(6)所得的总碱浸膏经硅胶柱色谱分离,用氯仿-甲醇进行梯度洗脱,获得若干个馏分;
(8)构建2,4-二硝基氯苯诱导的正常人皮肤成纤维细胞(NHDF)损伤模型,评价步骤(7)所得的各馏分的保护活性;
(9)将步骤(8)中具有最佳保护活性的馏分经大孔树脂分离,进一步用乙醇-水洗脱,进一步得到不同部位的若干个馏分;
(10)基于前述构建的2,4-二硝基氯苯诱导的正常人皮肤成纤维细胞(NHDF)损伤模型,评价步骤(9)所得的各馏分的保护活性;
(11)将步骤(10)评价出的、具有最佳保护活性的部位的馏分作为苦参提取物;
(12)取步骤(11)中所得的苦参提取物,经反复硅胶色谱柱、氯仿-甲醇梯度洗脱,再经ODS柱色谱及HPLC分离纯化,得到如式4、式5以及式6所示的苦参生物碱类化合物:
其中,步骤(2)中所采用的乙醇-水为25%-35%的乙醇;步骤(4)中所述的弱酸性为pH=5.5-6.5;步骤(6)中所述的弱碱性为pH=9.5-10.5。
2.如权利要求1所述的制备方法,其中,在步骤(12)中还分离得到如式1、式2、式3以及式7所示的苦参生物碱类化合物:
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