CN110642821B - 酢浆草中一种化合物及其提取分离方法及应用 - Google Patents
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Abstract
本发明公开了一种酢浆草中一种化合物及其提取分离方法及应用,该化合物为活性木脂素类化合物,结构式如式Ⅰ所示:4‑(3′‑甲氧基‑4′‑羟基)‑苯基‑6,7‑二甲氧基‑2,3‑萘内酯(Ⅰ);提取分离方法包括以下步骤:(1)取酢浆草原料,用乙醇提取,浓缩,得到浸膏;(2)将浸膏用上D‑101大孔吸附树脂,依次用水、50%EtOH、95%依次洗脱,得到若干组分;(3)将组分上样于柱色谱Ⅰ,用洗脱剂Ⅰ进行处理,取亚组分;(4)将亚组分用洗脱剂Ⅱ处理后,上样于柱色谱Ⅱ,再用洗脱剂Ⅲ处理,纯化得到活性木脂素类化合物。这种化合物能在制备抗宫颈癌、胃癌、肝癌、结肠癌、乳腺癌的药品和保健品方面得到广泛的应用。
Description
技术领域
本发明涉及中药提取、分离领域,尤其涉及从酢浆草药材中提取、分离和鉴别出的新化合物及其提取分离方法及应用。
背景技术
酢浆草Oxaliscorniculata L.为酢浆草科(Oxalidaceae)植物酢浆草的新鲜或干燥全草,苗药又名Suaib mib mib(秋咪咪),是贵州省少数民族常用药材,收载于《贵州省中药材、民族药材质量标准》(2003年版)。酢浆草在世界各地皆有分布,在中国,主要分布于华北、华中、华南及西南地区。
酢浆草的药用价值极高,在民间单方和复方均有应用。目前以酢浆草为原料的制剂有骨康胶囊、天胡荽愈肝片、妇炎消片、骨康片、肿痛舒喷雾剂等,主要用于强筋健骨、抗炎止痛、跌打损伤、活血化瘀等。现代药理学及临床研究表明,酢浆草不仅具有抗炎杀菌、活血化瘀、治疗跌打损伤的功效,还具有抗肿瘤、抗氧化、保肝等活性。经系统的文献调研发现,酢浆草含有多类化学成分,包括黄酮类(主要为黄酮及其苷类)、酚酸类、苯丙素类(主要为苯丙酸类)、生物碱类、萜类及其他类成分。
近年来,对酢浆草的生物活性研究发现,其提取物也具有一定的体内外抗肿瘤活性,因此,结合酢浆草化学成分研究,以期发现活性突出的化合物并进一步开发为抗肿瘤药物,在医学上是十分有前景的。
发明内容
针对现有技术存在的问题,本发明的目的是提供一种新化合物及其提取分离方法及应用,具体为从酢浆草中提取的新化合物,同时提供一种针对本发明新化合物的简便、快速的提取分离方法,以及在制备抗宫颈癌、胃癌、肝癌、结肠癌、乳腺癌等药物或保健品中的应用前景。
本发明的技术方案:
首先,本发明提出了一种酢浆草中一种新化合物,该新化合物为活性木脂素类化合物,该新化合物结构式如式Ⅰ所示:
4-(3′-甲氧基-4′-羟基)-苯基-6,7-二甲氧基-2,3-萘内酯(Ⅰ),结构式为:
该新化合物的提取分离方法,包括以下步骤:
(1)取酢浆草原料,用乙醇提取,浓缩,得到浸膏;
(2)将浸膏用上D-101大孔吸附树脂,依次用水、50%EtOH、95%依次洗脱,得到若干组分;
(3)将组分上样于柱色谱Ⅰ,用洗脱剂Ⅰ进行处理,取亚组分;
(4)将亚组分用洗脱剂Ⅱ处理后,上样于柱色谱Ⅱ,再用洗脱剂Ⅲ处理,纯化得到活性木脂素类化合物。
其中:
步骤(1)中的所述酢浆草原料经过干燥和粉碎处理后使用。
步骤(1)中使用浓度至少75%的乙醇提取。
步骤(3)中所述柱色谱Ⅰ为硅胶柱色谱。
步骤(3)中所述洗脱剂Ⅰ为氯仿-甲醇。
步骤(4)中所述柱色谱Ⅱ为Sephadex LH-20。
步骤(4)中所述洗脱剂Ⅱ为石油醚-丙酮,或者为氯仿-甲醇少量冰醋酸。
步骤(4)中所述洗脱剂Ⅲ为甲醇。
本发明还提出这种活性木脂素类化合物在制备抗宫颈癌、胃癌、肝癌、结肠癌或乳腺癌的药品或保健品中的应用。
本发明的有益效果是:本发明的方法,可以从酢浆草中提取分离得到一种新的活性木脂素类化合物,这种化合物能在制备抗宫颈癌、胃癌、肝癌、结肠癌、乳腺癌的药品和保健品方面得到广泛的应用。因此研究酢浆草的化学成分对明确其物质基础具有重要意义。
附图说明
图1为本发明新化合物Ⅰ的HR-ESI-MS(正模式);
图2为本发明新化合物Ⅰ的HR-ESI-MS(负模式);
图3为本发明新化合物Ⅰ的1H-NMR光谱图;
图4为本发明新化合物Ⅰ的13C-NMR光谱图;
图5为本发明新化合物Ⅰ的核磁共振HMQC光谱图;
图6为本发明新化合物Ⅰ的核磁共振HMBC光谱图。
具体实施方式
下面结合具体实施方式对本发明的技术方案作进一步说明。下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。下述方法中所涉及配料或材料,如无特殊说明,均为商业途径可获得。相关实验方法中如无特殊说明的均为本技术领域现有常规方法。其中的数值或数值比例,如无标注,均指质量数值或质量比例。
实施例1:
步骤1、干燥的酢浆草全株,切碎得粗粉15kg,用75%乙醇加热回流提取3次(1.5小时/次),合并提取液后减压回收溶剂并继续浓缩,得浸膏(2.1kg),醇提液滤过,合并滤液回收乙醇,直接加热浓缩不断加水挥乙醇至浓缩到无醇味;
步骤2、将浸膏经D-101大孔吸附树脂,用水(2.5倍柱体积)、50%EtOH(3倍柱体积)、95%EtOH(6倍柱体积)依次洗脱。分段收集,减压浓缩至浸膏得水段(1.8kg)、50%乙醇段(110g)、95%乙醇段(20g);
步骤3、合并50%乙醇段、95%乙醇段,再经正相硅胶柱层析,以氯仿-甲醇(50:1→0:1)进行梯度洗脱,分段收集,各段进行TLC检测合并后浓缩,得到12个组分(Fr.1-12)将合并后的洗脱部位经减压浓缩至干,备用;
步骤4、Fr.9过正相硅胶柱,以氯仿-甲醇(9:1→5:5,加少量冰醋酸)进行梯度洗脱,TLC检测合并、浓缩,得到8个组分(Fr.9.1-9.8),Fr.9.2.3过Sephadex LH-20凝胶柱(甲醇),得化合物;
化合物Ⅰ:淡黄色无定型固体。HR-ESI-MS m/z:367.1204[M+H]+(计算值:367.1182),365.1107[M–H]–(计算值:365.1025),分子式为C21H18O6;
1H-NMR(CDCl3,400MHz)显示了芳基萘内脂型木脂素所特有的质子信号,其中δ(5.23,1H,d,J=14.8Hz),(5.19,1H,d,J=14.8Hz)为内酯环亚甲基上的两个质子信号,根据这一特征信号可以判断该化合物的羰基与苯环于萘环异侧,即化合物的基本母核是4-苯代萘内酯;δ(7.08,1H,d,J=8.4Hz),(6.89,1H,dd,J=9.2,1.6Hz),(6.85,1H,d,J=1.6Hz)为一组ABX耦合质子信号,提示化合物中存在一个1,3,4-三取代苯片段;δ(8.28,1H,s),(7.28,1H,s),(7.10,1H,s)为萘环上的三个孤立质子信号,其中除去1位上的质子信号δ(8.28,1H,s),剩余质子信号δ(7.28,1H,s),(7.10,1H,s)提示化合物中存在1,2,4,5-四取代苯环片段;δ(4.03,3H,s),(3.89,3H,s),(3.82,3H,s)为三个甲氧基的质子信号。13C-NMR(CDCl3,100MHz)显示了21个碳信号,其中包含1个羰基碳信号δ171.7;16个芳香碳信号δ151.9,150.0,146.9,145.6,137.9,132.3,131.7,129.9,128.0,124.0,122.3,121.3,115.1,111.4,107.6,104.1;1个亚甲基碳信号δ69.6;3个甲氧基碳信号δ56.1,56.1,55.9。通过HMQC和HMBC谱对该化合物的1H-NMR、13C-NMR谱数据进行了归属。HMBC谱中,7-OCH3(δ3.82,3H,s)和H-8(δ7.28,1H,s)与C-7(δ151.9)远程相关,3′-OCH3(δ3.89,3H,s)和H-5′(δ7.08,1H,d,J=8.4Hz)与C-3′(δ146.9)远程相关,6-OCH3(δ4.03,3H,s)和H-5(δ7.10,1H,s)与C-6(δ150.0)远程相关,H-6′(6.89,1H,dd,J=9.2,1.6Hz)与C-4′(δ145.6)远程相关,提示取代基的位置。
经鉴定,该化合物Ⅰ为4-(3′-甲氧基-4′-羟基)-苯基-6,7-二甲氧基-2,3-萘内酯,其核磁共振谱图如表1所示。
表1:新化合物Ⅰ的1H-NMR(CDCl3,400MHz)和13C-NMR(CDCl3,100MHz)及HMBC数据
实施例2:
步骤1、干燥的酢浆草全株,切碎得粗粉15kg,用75%乙醇加热回流提取3次(1.5小时/次),合并提取液后减压回收溶剂并继续浓缩,得浸膏(2.1kg),醇提液滤过,合并滤液回收乙醇,直接加热浓缩不断加水挥乙醇至浓缩到无醇味;
步骤2、将浸膏经D-101大孔吸附树脂,用水(2.5倍柱体积)、50%EtOH(3倍柱体积)、95%EtOH(6倍柱体积)依次洗脱。分段收集,减压浓缩至浸膏得水段(1.8kg)、50%乙醇段(110g)、95%乙醇段(20g);
步骤3、合并50%乙醇段、95%乙醇段,再经正相硅胶柱层析,以氯仿-甲醇(50:1→0:1)进行梯度洗脱,分段收集,各段进行TLC检测合并后浓缩,得到12个组分(Fr.1-12)将合并后的洗脱部位经减压浓缩至干,备用;
步骤4、Fr.9过正相硅胶柱,以氯仿-甲醇(9:1→5:5,加少量冰醋酸)进行梯度洗脱,TLC检测合并、浓缩,得到8个组分(Fr.9.1-9.8),Fr.9.2.3过Sephadex LH-20凝胶柱(甲醇),得化合物;
为检测化合物Ⅰ对不同肿瘤细胞增殖的影响,现做以下抗肿瘤实验;
抗肿瘤实验的实验原理为:MTT全称
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazoliumbromide,又名四甲基偶氮唑蓝,商品名为噻唑蓝,是一种黄色染料。MTT法又称MTT比色法,是一种检测细胞存活和生长的方法。其检测原理为活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT还原为水不溶性的蓝紫色结晶甲臜(Formazan)并沉积在细胞中,而死细胞无此功能。二甲基亚砜(DMSO)能溶解细胞中的甲臜,用酶标仪在490nm波长处测定其光吸收值,可间接反映活细胞数量。在一定细胞数范围内,MTT结晶形成的量与细胞数成正比;
步骤1、在胎牛血清中培养不同种类的癌细胞,其中不同癌细胞与编号的对应关系如表2所示;
不同人的癌细胞:人肺癌细胞株A549;人肝癌细胞株HepG-2;人肝癌细胞株SMMC-7721;人乳腺癌细胞株MCF-7;人结肠癌细胞HCT-15;人神经胶质瘤细胞U251。
步骤2、将实验分为实验组(化合物Ⅰ)和对照组,其中对照组为顺铂,不同组的药物均设置有5个浓度梯度的加药,以上每组均设有5个复孔;
步骤3、收集对数生长期细胞,调整细胞浓度约为4-5×104个/mL,每孔100μL接种于96孔板中,放置培养箱中培养24h使其贴壁,加入含不同药物浓度的含药培养液100μL,并设置空白组(完全培养液);每组设计6个复孔。置于细胞培养箱培养24h,然后吸掉旧培养液,每孔加入20μL浓度为5mg/mL MTT,继续培养4h后,小心吸弃培养上清液,每孔加入150μL的DMSO溶解结晶甲瓒,在室温下震摇10min,等结晶完全溶解后,用酶标仪在490nm处检测光密度(OD,optical density)。最后根据OD值计算细胞抑制率。细胞抑制率IR(%)=[(OD值空白组-OD值实验组)/OD值对照组]×100%。
表2化合物Ⅰ对肿瘤细胞活性的影响
其中,IC50(μM)为增殖抑制率为50%时化合物Ⅰ的浓度,用于表示抗肿瘤活性;顺铂为阳性对照药。
如表2所示,化合物Ⅰ对人肝癌细胞SMMC-7721、人肺癌A549细胞,人结肠癌细胞HCT-15细胞以及人乳腺癌细胞MCF-7细胞都具较显著的抑制活性。
其中,IC50(μM)为增殖抑制率为50%时化合物Ⅰ的浓度,用于表示抗肿瘤活性;顺铂为阳性对照药合物Ⅰ对人肝癌细胞SMMC-7721、人肺癌A549细胞,人结肠癌细胞HCT-15细胞以及人乳腺癌细胞MCF-7细胞都具较显著的抑制活性。
以上对本发明的实施方式作了详细说明,但本发明不限于所描述的实施方式。对于本领域的技术人员而言,在不脱离本发明原理和精神的情况下,对这些实施方式进行多种变化、修改、替换和变型,仍落入本发明的保护范围内。
Claims (1)
1.酢浆草中的活性木脂素类化合物在制备抗肝癌、肺癌、结肠癌或乳腺癌的药品中的应用,其特征在于:所述的活性木脂素类化合物对MMC-7721、MCF-7、HCT-15及A549具有抑制活性的作用;所述的活性木脂素类化合物的结构式为:
所述活性木质素类化合物的提取分离方法包括以下步骤:
(1)取酢浆草原料,用乙醇提取,浓缩,得到浸膏;
(2)将浸膏用上D-101大孔吸附树脂,依次用水、50%EtOH、95%EtOH洗脱,得到若干组分;
(3)将组分上样于柱色谱Ⅰ,用洗脱剂Ⅰ进行处理,取亚组分;
(4)将亚组分用洗脱剂Ⅱ处理后,上样于柱色谱Ⅱ,再用洗脱剂Ⅲ处理,纯化得到活性木脂素类化合物;
其中:
步骤(1)中的所述酢浆草原料经过干燥和粉碎处理后使用;
步骤(1)中使用浓度至少75%的乙醇提取;
步骤(3)中所述柱色谱Ⅰ为硅胶柱色谱;
步骤(3)中所述洗脱剂Ⅰ为氯仿-甲醇;
步骤(4)中所述柱色谱Ⅱ为Sephadex LH-20;
步骤(4)中所述洗脱剂Ⅱ为石油醚-丙酮,或者为氯仿-甲醇少量冰醋酸;
步骤(4)中所述洗脱剂Ⅲ为甲醇。
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