CN114853713B - 水母雪莲中七种倍半萜类化合物及其提取分离方法和应用 - Google Patents
水母雪莲中七种倍半萜类化合物及其提取分离方法和应用 Download PDFInfo
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Abstract
本发明公开了水母雪莲中七种倍半萜类化合物及其提取分离方法和应用,属于中药提取、分离技术领域。本发明七种倍半萜类化合物分子式分别为C15H18O3、C15H18O4、C15H20O3、C19H30O7、C11H16O3、C11H18O4和C10H16O3。本发明采用乙醇回流提取、MCI柱层析、薄层层析、硅胶柱层析、SephadexLH‑20柱层析、高效液相色谱进行分离纯化与制备,采用ESIMS、1H‑NMR、13C‑NMR、HQBC、HMBC及2DNMR鉴定结构。本发明倍半萜类化合物及其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,用于制备抗肿瘤作用的药物。
Description
技术领域
本发明属于中药提取、分离技术领域,具体涉及水母雪莲中七种倍半萜类化合物及其提取分离方法和应用。
背景技术
水母雪莲(Saussurea medusa Maxim),是一种著名的传统藏药,被称为“雪莲”,用于治疗头部创伤、炭疽、发热刺痛、妇科疾病、类风湿性关节炎和中风。现代药理学研究表明,雪莲中主要含有木脂素、酚酸、类黄酮、萜类等化合物,在抗氧化、抗炎、降血糖、抑菌、抗癌等方面具有明显作用。
研究表明,水母雪莲中含有的倍半萜类化合物可明显抑制癌细胞的增殖,然而从水母雪莲中只有少数倍半萜被发现,目前仅仅从雪莲中分离鉴定出16个倍半萜类化合物,且分离出的倍半萜类化合物大多数是已知的,结构新颖性较低。因此,从水母雪莲中分离鉴定倍半萜类化合物并将其应用于抗癌、药物合成等具有重要的应用价值。
发明内容
为解决现有技术中的上述问题,本发明提供了水母雪莲中七种倍半萜类化合物及其提取分离方法和应用。
为实现上述目的,本发明提供了如下技术方案:
本发明技术方案之一:水母雪莲中七种倍半萜类化合物,结构式如式1~7所示
式4中,R为-O-β-D-glc或-OH中的一种。
进一步地,上述一类倍半萜类化合物分子式分别为C15H18O3、C15H18O4、C15H20O3、C19H30O7、C11H16O3、C11H18O4和C10H16O3,将其命名为SaussurenoidA,SaussurenoidB,Saussurenoid C,Saussurenoid D,Saussurenoid E,Saussurenoid F和Saussurenoid G。
本发明技术方案之二:上述水母雪莲中七种倍半萜类化合物的提取分离方法,包括以下步骤:
(1)将水母雪莲用乙醇回流提取,将提取液浓缩制得药液,将药液悬浮在水上,依次用石油醚、乙酸乙酯和正丁醇萃取,得到石油醚部位、乙酸乙酯部位和正丁醇部位;
(2)将步骤(1)得到的乙酸乙酯部位上MCI凝胶柱,用甲醇-水溶液梯度洗脱,收集组分,用薄层层析法合并相似组分,依次得到组分F1~F7;
(3)将步骤(2)得到的组分F2,F4,F5分别上硅胶柱,用二氯甲烷-甲醇溶液梯度洗脱,依次得到组分F2a~2g、F4a~F4g、F5a~F5g;
(4)将步骤(3)得到的组分F5b、F5d分别上Sephadex LH-20柱,用甲醇-水溶液梯度洗脱,依次得到组分F5b1~F5b4、F5d1~F5d4;
(5)将步骤(3)、(4)得到的组分F2a、F4e、F4d、F5b2,F5d1经高效液相色谱分离,得到所述倍半萜类化合物。
进一步地,所述乙醇的浓度为50%~95%,所述乙醇与所述水母雪莲的液固比(8~30mL):1g;所述回流提取的次数为3次,每次回流提取的时间为2h。
进一步地,步骤(2)中所述甲醇-水溶液中甲醇的体积比为0%~100%,所述梯度洗脱甲醇的体积比依次为0%、10%、30%、50%、70%、90%,100%。
进一步地,步骤(3)中所述二氯甲烷-甲醇溶液中二氯甲烷与甲醇的体积比为400:1~10:1,所述梯度洗脱二氯甲烷与甲醇的体积比依次为400:1、200:1、100:1、50:1,30:1,10:1,1:1。
进一步地,步骤(4)中所述甲醇-水溶液中甲醇的体积比为0%~100%,所述梯度洗脱甲醇的体积比依次为0%、10%、30%、50%、70%、90%,100%。
进一步地,步骤(5)中所述高效液相色谱分离是以体积分数为55%甲醇水溶液为流动相进行等度洗脱。
本发明技术方案之三:上述的水母雪莲中七种倍半萜类化合物在制备抗肿瘤药物中的应用。
与现有技术相比,本发明具有以下有益效果:
(1)本发明中由水母雪莲提取的倍半萜类化合物及其提取方法未被现有技术记载;本发明提取分离方法简便、快速,分得的化合物纯度高。
(2)本发明水母雪莲中七种倍半萜类化合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,用于制备抗肿瘤作用的药物。本发明的倍半萜类化合物和衍生物可应用于天然产物中药新药的开发,具有广阔的应用开发前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明化合物1的1H-NMR光谱图
图2为本发明化合物1的13C-NMR光谱图
图3为本发明化合物1的HSQCNMR光谱图
图4为本发明化合物1的HMBCNMR光谱图
图5为本发明化合物1的1H–1HCOSY光谱图
图6为本发明化合物1的ROESY光谱图
图7为本发明化合物1的(+)-ESIMS光谱图
图8为本发明化合物2的1HNMR光谱图
图9为本发明化合物2的13CNMR光谱图
图10为本发明化合物2的HSQC光谱图
图11为本发明化合物2的HMBC光谱图
图12为本发明化合物2的1H–1HCOSY光谱图
图13为本发明化合物2的ROESY光谱图
图14为本发明化合物2的(+)-ESIMS光谱图
图15为本发明化合物3的1HNMR光谱图
图16为本发明化合物3的13CNMR光谱图
图17为本发明化合物3的HSQC光谱图
图18为本发明化合物3的HMBC光谱图
图19为本发明化合物3的1H–1HCOSY光谱图
图20为本发明化合物3的ROESY光谱图
图21为本发明化合物3的(+)-ESIMS光谱图
图22为本发明化合物3的(-)-ESIMS光谱图
图23为本发明化合物4的1HNMR光谱图
图24为本发明化合物4的13CNMR光谱图
图25为本发明化合物4的HSQC光谱图
图26为本发明化合物4的HMBC光谱图
图27为本发明化合物4的1H–1HCOSY光谱图
图28为本发明化合物4的ROESY光谱图
图29为本发明化合物4的(-)-ESIMS光谱图
图30为本发明化合物5的1HNMR光谱图
图31为本发明化合物5的13CNMR光谱图
图32为本发明化合物5的HSQC光谱图
图33为本发明化合物5的HMBC光谱图
图34为本发明化合物5的1H–1HCOSY光谱图
图35为本发明化合物5的(+)-ESIMS光谱图
图36为本发明化合物5的(-)-ESIMS光谱图
图37为本发明化合物6的1HNMR光谱图
图38为本发明化合物6的13CNMR光谱图
图39为本发明化合物6的HSQC光谱图
图40为本发明化合物6的HMBC光谱图
图41为本发明化合物6的1H–1HCOSY光谱图
图42为本发明化合物6的ROESY光谱图
图43为本发明化合物6的(-)-ESIMS光谱图
图44为本发明化合物7的1HNMR光谱图
图45为本发明化合物7的13CNMR光谱图
图46为本发明化合物7的HSQC光谱图
图47为本发明化合物7的HMBC光谱图
图48为本发明化合物7的1H–1HCOSY光谱图
图49为本发明化合物7的ROESY光谱图
图50为本发明化合物7的(+)-ESIMS光谱图
图51为本发明化合物7的(-)-ESIMS光谱图
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本发明说明书和实施例仅是示例性的。
实施例
水母雪莲中倍半萜类化合物的提取
(1)取水母雪莲干燥药材15kg,加入150kg体积分数为95%的乙醇,回流提取3次,每次2h,将提取液过滤浓缩,减压回收乙醇,制得药液;
(2)将步骤(1)制得的药液悬浮在水中,依次用石油醚、乙酸乙酯和正丁醇萃取,得到石油醚部位、乙酸乙酯部位和正丁醇部位;
(3)将步骤(2)所得的乙酸乙酯部位上MCI凝胶柱,用甲醇的体积比为0%~100%的甲醇-水溶液进行梯度洗脱(甲醇的体积比依次为0%、10%、30%、50%、70%、90%,100%),收集洗脱组分,并用薄层层析法合并相似组分,得到组分F1~F7;
(4)将步骤(3)得到的组分F2,F4,F5分别上硅胶柱,用二氯甲烷与甲醇的体积比为400:1~10:1的二氯甲烷-甲醇溶液进行梯度洗脱(二氯甲烷与甲醇的体积比依次为400:1、200:1、100:1、50:1,30:1,10:1,1:1。),收集洗脱组分,依次得到组分F2a~2g、F4a~F4g、F5a~F5g;
(5)将步骤(4)得到的组分F5b、F5d分别上Sephadex LH-20柱,用甲醇的体积比为0%~100%的甲醇-水溶液进行梯度洗脱(甲醇的体积比依次为0%、10%、30%、50%、70%、90%,100%),收集洗脱组分,依次得到组分F5b1~F5b4、F5d1~F5d4。
(6)将步骤(4)、(5)得到的组分F2a、F4e、F4d、F5b2,F5d1利用高效液相色谱以55vol%甲醇水溶液为流动相进行等度洗脱,得到化合物1(5mg),化合物2(3mg),化合物3(5mg),化合物4(12mg),化合物5(5mg),化合物6(2mg)和化合物7(2mg)。
对上述制备得到的七种倍半萜类化合物采用ESIMS、1H-NMR、13C-NMR、HQBC、HMBC及2DNMR进行结构鉴定,结果如图1~51所示。其中图1~7为化合物1的谱图;图8~14为化合物2的谱图;图15~22为化合物3的谱图;图23~29为化合物4的谱图;图30~36为化合物5的谱图;图37~43为化合物6的谱图;图44~51为化合物7的谱图。
化合物1、2、7的1H-NMR与13C-NMR在CD3OD中,化合物3~6的1H-NMR与13C-NMR在CDCl3中,核磁数据见表1;
表1
本发明七种新化合物1~7的结构鉴定:
1:Saussurenoid A,白色无定形粉末; 1H-NMR与13C-NMR见表1;IR(KBr)νmax 3445,2962,1714,1584,1486,1384,1248,1040,925cm-1;UV(MeOH)λmax(logε)206(3.78),240(3.14),298(2.85)nm;CD(MeOH)λ(Δε)238(+13.86),258(-21.44),297(-7.22)nm;(+)-ESIMS m/z247.2[M+H]+;(+)-HRESIMS m/z 247.1326[M+H]+(calcd for C15H19O3,247.1329)。
2:Saussurenoid B,白色无定形粉末;[α]25D-41.3(c 0.31in MeOH);1H-NMR与13C-NMR见表1;IR(KBr)νmax3409,2961,2927,2870,1707,1584,1458,1384,1369,1248,1070,1041,1012cm-1;UV(MeOH)λmax(logε)206(3.68),240(3.09),295(2.70)nm;CD(MeOH)λ(Δε)240(+12.22),261(-16.26),297(-7.05)nm;(+)-ESIMS m/z 285.2[M+Na]+;(+)-HRESIMSm/z 263.1277[M+H]+(calcd for C15H19O4,263.1278)。
3:Saussurenoid C,白色无定形粉末;[α]25D-57.1(c 0.50in MeOH);1H-NMR与13C-NMR见表1;IR(KBr)νmax2961,1779,1747,1660,1444,1379,1173,1129,1025cm-1;UV(MeOH)λmax(logε)209(3.22),239(3.09);CD(MeOH)λ(Δε)210(+5.09),233(-9.91)nm;(+)-ESIMSm/z 266.2[M+NH4]+;(-)-ESIMS m/z 283.3[M+Cl]-;(+)-HRESIMS m/z 249.1484[M+H]+(calcd for C15H21O3,249.1485)。
4:Saussurenoid D,白色无定形粉末;[α]25D-14.7(c 0.30in MeOH);1H-NMR与13C-NMR见表1;IR(KBr)νmax3386,2957,2926,2856,1663,1609,1452,1420,1362,1259,1161,1078,1040cm-1;UV(MeOH)λmax(logε)195(3.68),221(3.36),283(3.25)nm;CD(MeOH)λ(Δε)208(-16.35),234(+3.58),281(+12.41)nm;(-)-ESIMS m/z 405.3[M+Cl]-;(+)-HRESIMSm/z 393.1888[M+Na]+(calcd for C19H30O7Na,393.1884)。
5:Saussurenoid E,淡黄色结晶(MeOH),mp 180-182℃;1H-NMR与13C-NMR见表1;IR(KBr)νmax3397,2963,2926,1732,1627,1516,1430,1383,1333,1250,1088,1035cm-1;UV(MeOH)λmax(logε)200(3.68),257(3.54),287(3.09)nm;(+)-ESIMS m/z 219.1[M+Na]+;(-)-ESIMS m/z 195.2,[M-H]-;(+)-HRESIMS m/z 219.0987[M+Na]+(calcd forC11H16O3Na,219.0992)。
6:Saussurenoid F,无色油状物;[α]25D+46.0(c 0.10in MeOH);1H-NMR与13C-NMR见表1;IR(KBr)νmax3389,2962,2926,2855,1743,1673,1456,1413,1384,1261,1087,1033cm-1;UV(MeOH)λmax(logε)248(3.69)nm;CD(MeOH)λ(Δε)215(-11.28),248(+33.89)nm;(-)-ESIMS m/z 249.1[M+Cl]-;(+)-HRESIMS m/z 237.1101[M+Na]+(calcd forC11H18O4Na,237.1097)。
7:Saussurenoid G,无色油状物;[α]25D-2.1(c 0.60in MeOH);1H-NMR与13C-NMR见表1;IR(KBr)νmax3378,2963,2928,2857,1704,1628,1447,1413,1261,1094,1036cm-1;UV(MeOH)λmax(logε)241(3.71)nm;CD(MeOH)λ(Δε)209(+12.76),243(-15.5)nm;(+)-ESIMSm/z 185.2[M+H]+;(-)-ESIMS m/z183.3[M-H]-;(+)-HRESIMS m/z 185.1169[M+H]+(calcdfor C10H17O3,185.1172)。
此外,由于上述谱图能够清晰地表现出新的七种倍半萜类化合物的结构,这也能证明提取的七种倍半萜类化合物纯度较高。
效果例
本效果例中胎牛血清购自美国Gibco公司;CCK-8试剂盒,阿霉素购自Meilun,中国大连;PBS购自北京中杉金桥生物技术有限公司;
抗肿瘤作用
(1)细胞培养:将A549(人肺腺癌细胞系)及HL-60(人早幼粒急性白血病细胞)置于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)CCK-8试剂法测定细胞活力:取对数生长期的A549及HL-60接种于96孔板(Falcon,CA)中,细胞密度为1×104个/mL,每孔90μL,温度37℃,5%CO2条件下培养过夜后,用不同浓度的受试化合物(阿霉素以及除化合物6外的七种倍半萜类化合物)处理,再生长72小时,每个浓度一式三份。暴露时间结束后,每孔加入10μLCCK-8,之后在培养箱中保持4h。在SpectraMax 190酶标仪上以450nm的波长测量每孔的光密度(OD)。
IC50值测试结果如表2所示,
表2
aData expressed as the mean±SD(n=3).bIA stands for inactive(definedas an inhibition rate<50%at 10μM)cPositive control.
由上表可知,化合物2对A549及HL-60显示出较好的活性,化合物3对A549显示出较好的活性。
以上所述,仅为本发明较佳的具体实施方式,本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围内。
Claims (4)
1.水母雪莲中两种倍半萜类化合物,其特征在于,结构式为
2.权利要求1所述的水母雪莲中两种倍半萜类化合物的提取分离方法,其特征在于,包括以下步骤:
(1)将水母雪莲用乙醇回流提取,将提取液浓缩制得药液,将药液悬浮在水上,依次用石油醚、乙酸乙酯和正丁醇萃取,得到石油醚部位、乙酸乙酯部位和正丁醇部位;
(2)将步骤(1)得到的乙酸乙酯部位上MCI凝胶柱,用甲醇-水溶液梯度洗脱,收集组分,用薄层层析法合并相似组分,依次得到组分F1~F7;
(3)将步骤(2)得到的组分F2,F4,F5分别上硅胶柱,用二氯甲烷-甲醇溶液梯度洗脱,依次得到组分F2a~2g、F4a~F4g、F5a~F5g;
(4)将步骤(3)得到的组分F5b、F5d分别上Sephadex LH-20柱,用甲醇-水溶液梯度洗脱,依次得到组分F5b1~F5b4、F5d1~F5d4;
(5)将步骤(3)、(4)得到的组分F2a、F4e、F4d、F5b2,F5d1经高效液相色谱分离,得到所述倍半萜类化合物;
所述倍半萜类化合物的结构式为
步骤(2)中所述甲醇-水溶液中甲醇的体积比为0%~100%,所述梯度洗脱甲醇的体积比依次为0%、10%、30%、50%、70%、90%,100%;
步骤(3)中所述二氯甲烷-甲醇溶液中二氯甲烷与甲醇的体积比为400:1~1:1,所述梯度洗脱二氯甲烷与甲醇的体积比依次为400:1、200:1、100:1、50:1,30:1,10:1,1:1;
步骤(4)中所述甲醇-水溶液中甲醇的体积比为0%~100%,所述梯度洗脱甲醇的体积比依次为0%、10%、30%、50%、70%、90%,100%;
步骤(5)中所述高效液相色谱分离是以体积分数为55%甲醇水溶液为流动相进行等度洗脱。
3.根据权利要求2所述的提取分离方法,其特征在于,步骤(1)中所述乙醇的浓度为50%~95%,所述乙醇与所述水母雪莲的液固比(8~30mL):1g;所述回流提取的次数为3次,每次回流提取的时间为2h。
4.权利要求1所述的水母雪莲中两种倍半萜类化合物在制备抗肿瘤药物中的应用,其特征在于,所述应用于制备抗肺腺癌、人早幼粒急性白血病肿瘤药物中,所述
应用于制备抗肺腺癌肿瘤药物中。
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