CN113968774B - 马齿苋中一种多芳基化合物及其提取分离方法 - Google Patents
马齿苋中一种多芳基化合物及其提取分离方法 Download PDFInfo
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- CN113968774B CN113968774B CN202111394329.7A CN202111394329A CN113968774B CN 113968774 B CN113968774 B CN 113968774B CN 202111394329 A CN202111394329 A CN 202111394329A CN 113968774 B CN113968774 B CN 113968774B
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
- C07C41/01—Preparation of ethers
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Abstract
本发明涉及中药提取、分离领域,尤其涉及从马齿苋中提取、分离和鉴别出的一种新的多芳基化合物及其提取分离方法。所述的多芳基化合物,分子式为C60H58O,1,1'‑oxybis(2‑((R)‑1‑phenylethyl)‑4,5‑bis((S)‑1‑phenylethyl)phenyl)。还提供上述多芳基化合物的提取分离方法,依次采用醇溶剂煎煮回流提取、大孔树脂柱层析、硅胶柱层析、ODS中压柱及羟丙基葡聚糖柱层析纯化、液相分离制备。其结构采用1H‑NMR、13C‑NMR及UHPLC‑ESI‑Q‑TOF‑MS等方法确定化合物结构。该化合物具有潜在的神经保护和抗胆碱酯酶等活性,并提供制备方法,为开发新药和开发新成分提供先导物和理论依据。
Description
技术领域
本发明涉及中药提取、分离领域,尤其涉及从马齿苋药材中提取、分离和鉴别出的多芳基化合物及其提取分离方法。
背景技术
马齿苋(Portulaca oleracea L.),又名长命菜、马苋菜,为马齿苋科植物。马齿苋性喜肥沃土壤,耐旱亦耐涝,生命力强,分布广泛,资源丰富,而以我国东北部的更为常见。马齿苋既可入药,又可食用,是我国卫生部划定的药食同源的野生植物之一。2020版《中华人民共和国药典》中收载马齿苋的干燥地上部分入药,具有清热解毒、凉血止血、止痢等功效,用于热毒血痢、痈肿疔疮、湿疹、丹毒、蛇虫咬伤、便血、痔血和崩漏下血等。
现代药理学研究表明,马齿苋具有降血脂、降血糖、抗炎、抗氧化、抗肿瘤、抗动脉粥样硬化和松弛或兴奋平滑肌及增强免疫力等功效。研究表明马齿苋中所含多种化学成分与其多样的药理作用息息相关,其主要化学成分包括:黄酮类、生物碱类、萜类、香豆素类、有机酸类、挥发油、多糖、氨基酸、各种色素类和矿物质类等。其中生物碱是马齿苋中的一大类活性成分,而酰胺类生物碱又占绝大多数。目前已报道的生物碱类成分有去甲肾上腺素、多巴胺、少量多巴、腺苷、尿嘧啶、腺嘌呤、N,N-二环己基脲、尿囊素、N-反式-阿魏酰基酪胺;还有环二肽生物碱和酰胺类生物碱:马齿苋酰胺A-I、K、L、N-S。
目前从马齿苋中分离出的化学成分大多数是已知的,且结构新颖性较低,因此,对马齿苋中化合物的开发和分离是亟待需要的。
发明内容
针对上述问题,本发明提供一种从马齿苋中提取的多芳基化合物,经研究发现本发明的多芳基化合物具有神经保护和抗胆碱酯酶的作用,同时提供一种针对本发明多芳基化合物的简便、快速、环保、纯度高的提取分离方法。
为了实现上述目的,本发明提供了如下技术方案。
本发明提供了一种从马齿苋药材中分离出的多芳基化合物,其特征在于,分子式分别为:C60H58O,并且根据结构命名为1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl),
。
本发明还提供给了一种从马齿苋药材中分离出的多芳基化合物的提取分离方法,所述提取分离方法的具体步骤为:
步骤1、取马齿苋干燥药材,采用醇溶剂煎煮回流提取,醇溶剂提液滤过,合并滤液蒸馏浓缩,放凉至室温,得药液备用;
步骤2、将步骤1中浓缩物经大孔树脂柱分离,采用乙醇-水梯度洗脱,70%乙醇部分蒸干后上硅胶柱,依次采用乙酸乙酯∶甲醇洗脱,减压回收乙酸乙酯至浸膏,得到乙酸乙酯提取物;
步骤3、将步骤2中所得物经预处理的ODS柱层析分离,用甲醇-水梯度洗脱,得到若干洗脱部位,经薄层色谱进行检测,显色,将显色部位减压浓缩至干,得到浓缩物备用;
步骤4、将步骤3中所得浓缩物经预处理的羟丙基葡聚糖凝胶层析分离,以甲醇等度洗脱,得到若干部位,经薄层色谱进行检测,显色,合并显色的洗脱部位,将合并的洗脱部位经减压浓缩至干,得浓缩物备用;
步骤5、将步骤4中所得浓缩物通过UHPLC分离制备,以乙腈-0.1%甲酸水作为流动相使用不同浓度进行等度洗脱,最终得到本发明所述的多芳基化合物。
进一步地,所述步骤1中醇溶剂为50%乙醇,提取次数为两次,每次提取回流2小时,乙醇的用量为药材的8~16倍。
进一步地,所述步骤2中所用乙醇-水洗脱程序为梯度洗脱,分别为30%,50%和70%乙醇。
进一步地,所述步骤2中乙酸乙酯和甲醇的体积比为1∶0、5∶1和2∶1。
进一步地,所述步骤3,4中所述ODS和羟丙基葡聚糖凝胶的预处理过程为甲醇浸泡过24小时,上柱,以初始流动相平衡。
进一步地,所述步骤3中甲醇和水的体积比为60∶40、70∶30、80∶20和90∶10梯度洗脱。
进一步地,所述步骤4中甲醇洗脱程序为等度洗脱。
进一步地,所述步骤5中乙腈-0.1%甲酸水的体积比为87∶13,得到所述的多芳基化合物1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl)。
本发明还提供了一种从马齿苋药材中分离出多芳基化合物的用途,其特征在于,所述用途可用于制备神经保护和抗胆碱酯酶的药物或保健品。
与现有技术相比本发明的有益效果。
本发明中所述马齿苋中多芳基化合物的分离和药理活性研究中未发现论文期刊有所报道;本发明提供来源于马齿苋的多芳基化合物及一种针对本发明化合物的提取分离方法,依次采用醇提取、大孔树脂柱层析、硅胶柱层析、ODS中压柱、羟丙基葡聚糖柱层析及HPLC进行分离纯化与制备,成功提取分离出的多芳基化合物,该方法操作步骤仅为五步,操作方法简便及快速,提取分离过程主要采用乙醇提取,工艺方法环保,且经该方法分离得到的化合物纯度均大于98%,此外经研究表明以上化合物具有神经保护和抗胆碱酯酶的作用,因此本发明多芳基化合物及其盐和衍生物可以作为其他化合物合成先导物,以及新药开发和药理活性研究的原料,亦可用于制备神经保护和抗胆碱酯酶的药物。
附图说明
图1为本发明多芳基化合物1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl)的1H-NMR光谱图。
图2为本发明多芳基化合物1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl)的13C-NMR光谱图。
图3为本发明多芳基化合物1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl)的DEPT光谱图。
图4为本发明多芳基化合物1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl)的核磁共振HMBC光谱图。
图5为本发明多芳基化合物1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl)的核磁共振1H-1HCOSY光谱图。
图6为本发明多芳基化合物1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl)的核磁共振HSQC光谱图。
图7为本发明多芳基化合物1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl)的核磁共振ROESY光谱图。
图8为本发明多芳基化合物1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl)的高分辨质谱图。
具体实施方式
以下实施例将有助于对本发明的了解,但这些实施例仅为了对本发明加以说明,本发明并不限于这些内容。在实施例中的操作方法均为本技术领域常规操作方法。
实施例1
本发明提供多芳基化合物,分子式为C60H58O命名为1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl),化学结构式为:
。
所述多芳基化合物根据结构命名为1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl),表1为该多芳基类化合物的核磁数据:1H-NMR与13C-NMR在DMSO中。
表1:本发明化合物1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl)的核磁数据
。
本发明多芳基类化合物1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl)的结构鉴定与推导。
1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl):化合物为白色粉末,易溶于甲醇。UHPLC-ESI-Q-TOF-MS给出m/z:795.4558[M-H]+(C60H59O+,计算值为795.4561)的准分子离子峰。结合1H-NMR,13C-NMR以及DEPT数据,推测该化合物可能的分子式为C60H58O,不饱和度为32。在1H-NMR和13C-NMR中可以看出,一些信号在1H-NMR和13C-NMR中都是加倍的。
13C-NMR谱和DEPT谱显示了13个碳原子信号,包括5个芳香次甲基,2个CH基团,1个CH3和其他5个季碳。根据1H-NMR谱,δH6.84(2H,dd,J=2.28,17.28)和δH6.93(2H,s)信号证明了1,2,4,5-四取代苯环的存在,另外三个氢信号δH7.18(12H,m)和δH7.22(12H,m)为AABB系统,δH7.12(6H,m)为芳香次甲基,根据积分情况推测其为三个一取代苯环。除不饱和氢原子外,在δH4.50(4H,m)和δH3.98(2H,m)处观察到两个次甲基信号,在δH1.45(18H,m)处观察到一个甲基信号。表1列出了1H-NMR(600MHz,MeOD)和13C-NMR(150MHz,MeOD)的数据。根据HMBC中,δH3.98(H-1′)与δC146.50(C-1′′,C-1′′′′和C-1′′′′′′)和δC127.31(C-2′′,C-2′′′′和C-2′′′′′′);δH1.45(H-2′,H-2′′′和H-2′′′′′)与δC146.50和δC43.68(C-1′);δH4.50(H-1′′′和H-1′′′′′)与δC146.50和δC127.31;δH1.45与δC146.50和δC36.86(C-1′′′和C-1′′′′′)相关,结合1H-1H COSY谱H-1′与H-2′;δH1.45与δH4.50相关,证明了三个苯乙基的存在。在HMBC中,H-3与C-1(δC149.07)和H-6与C-1的相关性,结合C-1的化学位移,表明1位与O相连。此外,H-1′与C-2(δC136.84)和C-3(δC123.82)相关,H-2′与C-2相关,H-1′′′与C-3和C-5(δC132.89)相关,H-2′′′与C-4(δC133.23)相关,H-1′′′′′与C-4和C-6(δC127.38)相关,H-2′′′′′与C-5相关,表明C-2,C-4和C-5分别与C-1′,C-1′′′和C-1′′′′′′相连。因为有些信号在1H-NMR和13C-NMR中都是双倍的,可以假设C-2通过氧连接到相同的结构上。并且在质谱图中找到了该结构的分子离子峰。根据以上信息,可确定此新化合物为上述结构。
根据以上信息,可确定此多芳基类化合物为上述结构。
本发明还提供上述多芳基类化合物的提取分离方法,具体步骤为。
步骤1:马齿苋干燥药材250kg,采用50%乙醇回流提取,50%乙醇用量为药材的8~16倍,回流提取两次,每次2h,减压回收乙醇,放凉至室温,得药液备用。
步骤2:将步骤1浓缩物经大孔树脂柱分离,采用乙醇-水(30∶70,50∶50,70∶30,v∶v)梯度洗脱,70%乙醇部分蒸干后上硅胶柱分离,用乙酸乙酯∶甲醇(1∶0,5∶1,2∶1,v∶v)梯度洗脱,其中硅胶为100~200目,40℃以下减压回收乙酸乙酯至浸膏,得到乙酸乙酯提取物。
步骤3:将步骤2中所得物再经预处理的ODS中压柱层析分离,其中填料粒度为20~40μm,用甲醇∶水(60∶40,70∶30,80∶20和90∶10,v∶v)梯度洗脱(加压,使流速为1mL/min,温度为室温),得到30个部位(即梯度洗脱得30个瓶,每瓶100mL),经薄层色谱进行检测,显色,将显色的20~25部位保留,50℃以下减压浓缩至干,备用。
步骤4:将步骤3中所得浓缩物再经预处理的羟丙基葡聚糖凝胶柱层析分离,用甲醇等度洗脱,得到20洗脱部位(即共得到20瓶,每瓶50mL),经薄层色谱进行检测,显色,将显色的7-10部位保留,50℃以下减压浓缩至干,备用,得到化合物。所述ODS与羟丙基葡聚糖凝胶的预处理过程为甲醇浸泡过24h,上柱,用甲醇洗至滴入水中无混浊,再以初始流动相平衡。
步骤5:将步骤4中所得化合物经UHPLC分离制备,以乙腈∶0.1%甲酸作为流动相分别用体积比为87∶13等度洗脱,检测波长为210nm和280nm,分离制备得到本发明的多芳基类化合物,归一法测定纯度均为≥98%。
实施例2本发明多芳基类化合物的神经保护作用。
1 主要材料。
1.1 药品和试剂:实验所用新生物碱化合物由上述方法制备,纯度为90~99%,精密称取,用DMSO稀释至下述各剂量组所需溶液。DMEM高糖培养基、胎牛血清(美国Hyclone公司);青霉素、链霉素(杭州四季青公司),磷酸盐缓冲液(PBS),(武汉博士德有限公司),ROS检测试剂盒(海门碧云天试剂公司)。
1.2 细胞株:人神经母细胞瘤细胞株(SH-SY5Y、IMR-32)(中科院上海细胞)。
1.3 分组:分为对照组、H2O2损伤模型组和实验组。
2 实验方法。
2.1 细胞培养,DMEM高糖培养基,加入l0%的胎牛血清,l%抗菌素(100U/mL青霉素和100μg/mL链霉素),置于37℃、5%CO2培养箱中培养。
2.2 MTT比色法测定细胞活力,上述三组分别取对数生长期SH-SY5Y细胞和IMR-32细胞接种于96孔培养板中,细胞密度为1×104个/mL,每孔100μL,温度37℃,5%CO2条件下培养过夜后,实验组加入不同浓度的本发明新生物碱化合物1,1'-oxybis(2-((R)-1-phenylethyl)-4,5-bis((S)-1-phenylethyl)phenyl)(5-40μM),孵育1h后向H2O2组和实验组分别加入终浓度为800μM/L的H2O2,另设调零组(含DMSO溶媒的培养液),每组设3个复孔,考察加入药物后对细胞的影响。上述各组细胞培养24h后,在各孔细胞中加入5 mg/mL MTT20μL,温度37℃,5%CO2条件下继续孵育4h后,终止培养,吸弃孔内液体,每孔加入100μL二甲基亚砜(DMSO),振荡10min,使细胞内结晶充分溶解,酶标仪450 nm波长处测定各孔吸光值(A)值,计算细胞存活率,细胞存活率=(AH2O2损伤-A空白)/(A对照-A空白)。
2.3 DCFH-DA法检测SH-SY5Y细胞和IMR-32细胞内ROS,各组细胞给予相应物质后孵育24h,孵育结束前30min,各孔加入DCFH-DA,使终浓度为10μmol/L,于37℃继续孵育30min,收集细胞,PBS洗2次,细胞计数,将各组细胞制成相同浓度的细胞悬液。取100μL细胞悬液检测荧光强度,激发波长485nm,发射波长538nm。以对照组荧光强度为100%,其余各组与对照组荧光强度相比较,计算胞内ROS变化。
2.4 INT显色反应法测定LDH的释放量,除上述对照组、H2O2损伤模型组和实验组外,另设立空白对照组(空白对照组不接种细胞),各组细胞加入相应物质培养24h,取各孔上清120μL至新的96孔板中,加60μL配好的LDH检测工作液,避光室温孵育30min,在490nm处用多功能酶标仪测定A值,计算相对于对照管的LDH释放量百分率。LDH释放率=(A给药-A空白)/(A对照-A空白)。
3 实验结果。
细胞相对存活率实验结果如表2所示。
表2:本发明对人神经母细胞瘤细胞株SH-SY5Y和IMR-32细胞相对存活率的影响
。
SH-SY5Y细胞和IMR-32细胞内ROS量检测结果如表3所示。
表3:本发明对人神经母细胞瘤细胞株SH-SY5Y和IMR-32细胞内ROS量的影响
。
SH-SY5Y细胞和IMR-32细胞内LDH释放的影响结果如表4所示。
表4:本发明对人神经母细胞瘤细胞株SH-SY5Y和IMR-32细胞内LDH释放的影响
。
实施例3本发明的化合物的抗胆碱酯酶作用。
1、主要材料。
1.1、药品和试剂:实验所用多芳基化合物由上述方法制备,纯度为90~99%,磷酸二氢钠、磷酸氢二钠(国药集团化学试剂有限公司),毒扁豆碱(瀚香生物科技),磷5,5’-二硫代双(2-硝基苯甲酸)(Dithiobisnitrobenzoic acid,DTNB,上海金穗生物科技有限公司),乙酰胆碱酯酶(AChE)和碘化硫代乙酰胆碱(Acetylthiocholine iodide,ATCI,大连美仑生物技术有限公司)。
1、分组:分为空白组、对照组和样品组。
2、实验方法。
2.1样品准备。
分别精密称取样品和毒扁豆碱0.11mg,分别以甲醇为溶剂,配置成2.5μM、5.0μM、10.0μM、20.0μM和40.0μM的五个梯度浓度。分别精密称取7.8005g的磷酸二氢钠和17.9070g的磷酸氢二钠,用蒸馏水定容至500mL,取26.5mL的磷酸二氢钠和473.5mL的磷酸氢二钠,配制成500mL的PBS(0.1M,pH=8.0);精密称取0.0594g的DTNB,加入10mL的PBS,配制成DTNB溶液(15mmol/L);精密称取0.01g的AChE,加入10mL的PBS,配制成AChE溶液(0.2U/mL);精密称取0.044g的ATCI,用蒸馏水定容至10mL,配制成ATCI溶液(15mmol/L)。
2.2改进的Ellman方法测定抗胆碱酯酶活性。
在96孔酶标板中依次加入140μL的PBS(0.1M,pH=8.0),10μL的DTNB(15mmol/L),15μL的AChE(0.2U/mL),20μL样品溶液。空白组实验用甲醇代替样品,阳性对照组实验用毒扁豆碱代替样品。37℃孵育10min后,加10μL的ATCI(15mmol/L)。20℃孵育10min后,用酶标仪在410nm下测定其吸光度值。根据下式计算抑制率:抑制率(%)=(空白组-样品组)/空白组×100%。
3、实验结果。
实验结果表明本发明多芳基化合物有抗胆碱酯酶作用。
实验结果如表5所示。
表5:本发明抗胆碱酯酶活性
。
综上所述,本发明提供特殊化合物及其提取分离方法,依次采用醇回流提取、大孔树脂柱层析、硅胶柱层析、ODS中压柱及羟丙基葡聚糖凝胶柱层析分离纯化,成功的分离得到化合物,该方法简便,快速,环保,且经该方法分离得到的化合物纯度较高,由于所得化合物化学结构独特,从常用中药马齿苋中提取出来,其具有神经保护和抗胆碱酯酶作用,因此本发明特殊化合物及其盐和衍生物可以作为天然产物开发中药新药,具有广阔的前景。
Claims (3)
1.一种从马齿苋药材中分离出的多芳基化合物,其特征在于,其化学结构式如下:
。
2.一种如权利要求1所述的从马齿苋药材中分离出的多芳基化合物的提取分离方法,其特征在于,所述提取分离方法的具体步骤为:
步骤1:马齿苋干燥药材250kg,采用50%乙醇回流提取,50%乙醇用量为药材的8~16倍,回流提取两次,每次2h,减压回收乙醇,放凉至室温,得药液备用;
步骤2、将步骤1浓缩物经大孔树脂柱分离,采用体积比为30∶70,50∶50,70∶30的乙醇-水梯度洗脱,70%乙醇部分蒸干后上硅胶柱分离,用体积比为1∶0,5∶1,2∶1的乙酸乙酯∶甲醇梯度洗脱,其中硅胶为100~200目,40℃以下减压回收乙酸乙酯至浸膏,得到乙酸乙酯提取物;
步骤3、将步骤2中所得物再经预处理的ODS中压柱层析分离,其中填料粒度为20~40μm,用体积比为60∶40,70∶30,80∶20和90∶10的甲醇∶水梯度洗脱,洗脱条件为:加压,使流速为1mL/min,温度为室温,得到30个部位,即梯度洗脱得30个瓶,每瓶100mL,经薄层色谱进行检测,显色,将显色的20~25部位保留,50℃以下减压浓缩至干,备用;
步骤4、将步骤3中所得浓缩物再经预处理的羟丙基葡聚糖凝胶柱层析分离,用甲醇等度洗脱,得到20洗脱部位,共得到20瓶,每瓶50mL,经薄层色谱进行检测,显色,将显色的7~10部位保留,50℃以下减压浓缩至干,备用;
步骤5、将步骤4中所得化合物经UHPLC分离制备,以体积比为87∶13的乙腈∶0.1%甲酸作为流动相等度洗脱,分离制备得到权利要求1所述的多芳基类化合物。
3.一种如权利要求1所述的从马齿苋药材中分离出多芳基化合物的用途,其特征在于,所述用途可用于制备神经保护和抗胆碱酯酶的药物。
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