CN101633609B - 一种从山莓叶中分离出的新化合物、制备方法及用途 - Google Patents
一种从山莓叶中分离出的新化合物、制备方法及用途 Download PDFInfo
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Abstract
本发明涉及一种从山莓叶中分离出的新化合物、制备方法及用途,化合物的化学名为:对映-贝壳杉-16α,17-二羟基-2酮[ent-kauran(5R,8S,9R,10R,13R,16R)-2-one-16α,17-diol],由山莓经过反复硅胶柱层析、薄层层析和结晶分离所得,其结构式有波谱解析和化学方法确定,经活性实验证明该化合物具有抗肿瘤作用。
Description
技术领域
本发明涉及从山莓叶中一种新化合物及其制备方法,以及其在抗肿瘤方面的作用。
背景技术
山莓(Rubus corchorifolius L.f)属蔷薇科悬钩子属中一种落叶灌木。多生长于向阳山坡、溪边、山谷、荒地和灌丛中,海拔300m~1500m。除东北、内蒙古、青海、新疆、西藏外,全国均有分布。福建全省各地均有山莓分布,野生资源蕴藏量相当大。我国利用山莓的历史悠久,在古书《本草拾遗》、《本草纲目》、《名医别录》、《食疗本草》中就有山莓药用价值的详细记载,其药用已有上千年历史。山莓果、根及叶均可入药。果以未成熟果入药,民间常作为覆盆子代用品。果性平味微甘、酸、温,具有涩精益肾、助阳明目之功效,能醒酒止渴、化痰解毒、收敛;主治醉酒、痛风、丹毒、烫火伤、肾虚、遗精、遗尿等症;根性平味微苦涩,具有活血化瘀、清热利湿、解毒敛疮的作用,主治风湿腰痛、咯血、崩漏、痔疮出血、痢疾、泻泄、闭经、痛经、跌打损伤、毒蛇咬伤、疮疡肿痛、湿疹、小儿疳积等症;叶性平微苦,具有清热利咽、解毒敛疮、消肿解毒的功效,主治咽喉肿痛、多发性脓肿、乳腺炎等症。其中地上部分(叶、幼果和茎)所含的香豆素化合物,如东茛菪内酯具有一定的镇痛、抗炎、祛痰和平喘的作用。在湘西、鄂西北山莓作为常用的苗族药,用于医治常见的多发病,如腹泻、目赤、酒精中毒等,疗效确切。
目前有关山莓的研究主要是集中在资源的开发利用方面,陈炳华利用其聚合果,用于食品加工;利用其鞣质作为化工原料;陈炳华等对山莓的营养成分及利用价值和茎中香豆素类成分进行了初步研究;周双德报道了山莓生态习性与叶的解剖结构。除此之外有关山莓化学成分方面的研究也有报道:安保礼等人从山莓叶中提取出茶多酚;陈炳华报道了山莓全株含鞣质、黄酮类化合物,地上部分(叶、茎、幼果)含香豆素化合物,根中含酚性成分和皂甙,从茎和叶中分离出一种香豆素成分;董振生对山莓叶的生物碱进行了提取和抑菌作用的研究。张敏从山莓叶中分离得到了两个新的二萜化合物。
发明内容
本发明的目的在于提供一种从山莓中分离出的新化合物,其化学名为:对映-贝壳杉-16α,17-二羟基-2酮[ent-kauran(5R,8S,9R,10R,13R,16R)-2-one-16α,17-diol],其结构经波谱解析和化学方法确定,活性实验证明该化合物具有抗肿瘤作用。
本发明提供的新化合物是从山莓叶中经过反复硅胶柱层析、薄层层析和结晶分离所得,化学名为:对映-贝壳杉-16α,17-二羟基-2酮[ent-kauran(5R,8S,9R,10R,13R,16R)-2-one-16α,17-diol],经波谱解析和化学方法确定其化学结构式为:
该化合物的制备方法包括以下步骤:
(1)称取粉碎的山莓叶,加入10倍体积80%的乙醇,搅匀,先超声提取30min,然后于室温下浸提48h,过滤,滤渣再重复提取一次,合并滤液,浓缩冻干得精粗提物浸膏液,备用。
(2)将精粗提物浸膏液分别用石油醚、氯仿、乙酸乙酯或正丁醇反复多次萃取,将萃取液浓缩,冷冻干,收集乙酸乙酯萃取物。
(3)将乙酸乙酯萃取物进行硅胶柱色谱分离,用正己烷与乙酸乙酯和乙酸乙酯与甲醇进行梯度洗脱,合并流出液,经TLC检测,显色,合并相同组份,经重结晶后得该化合物。
步骤(3)中,进行梯度洗脱时,正己烷与乙酸乙酯的体积比依次为:100∶0,100∶50,乙酸乙酯与甲醇的体积比依次为100∶0,80∶1,40∶1,10∶1,5∶1,1∶1,0∶1。
该化合物作为制备抗肿瘤药物的用途。
该化合物作为制备保健品的用途。
该化合物作为制备药物组分之一的用途。
本发明提供的新化合物的结构鉴定方法为:
1、化合物的理化数据
(1)白色针状晶体,M.p.150~152℃,旋光度:-23.8°:全波段扫描λmax=215nm(甲醇),用氯仿∶甲醇=20∶1,石油醚∶乙酸乙酯=2∶5,环己烷∶丙酮=3∶1三种不同溶剂系统上行展开,1%香草醛硫酸溶液显为单一红色斑点。在氯仿∶甲醇=20∶1系统中Rf=0.38,石油醚∶乙酸乙酯=2∶5系统中Rf=0.398,环己烷∶丙酮=3∶1系统中Rf=0.25。
(2)IR数据(溴化钾压片,cm-1):3540,3297,2936,2863,1698,1670,1469,1445,1368,1272,1211,1154,1068,1024,994,964,918,878,815。解析:3540,3297cm-1(强,宽),说明分子中含有羟基;1698cm-1(强,第一强峰)说明分子中含有羰基。
2、分子式的确定
APCI-MS给出分子量为320。在1H-NMR,中共有32个氢信号,HMQC中显示共有30个连碳氢,因此有两个活泼质子,对比碳谱数据,81.7ppm,66.2ppm,应该为连氧碳信号,因此分子中有两个羟基,而211ppm,DEPT显示为S信号,说明为一酮羰基,所以分子中共有20个碳原子,32个氢,3个氧,分子式为C20H32O3,分子量为320,Ω=5。
3、结构式的确定
分析该化合物的碳谱(COM,DEPT),20个碳原子,发现其碳谱数据与对映贝壳杉烷型二萜数据吻合,且在前面对该植物研究中发现了多个对映贝壳杉烷型二萜类成分。对映-16β,17-贝壳杉烷的原子编号如下:
在该类二萜碳谱数据对比研究中,发现对于对映-16α,17-二羟基贝壳杉烷,第13-17号碳化学位移信号,大概范围为:46,38,54,81,66(ppm)左右,而对于对映-16β,17-二羟基贝壳杉烷,第13-17号碳化学位移信号,大概范围为:42,38,53,80,70(ppm)左右,两者主要对于17号碳化学位移差距明显,而该化合物的此五个碳化学位移分别为:52.8,81.7,66.2(ppm)与对映-16β,17-二羟基贝壳杉烷吻合,故推测16,17位有两个羟基取代,其中16为α构型。进一步分析该化合物HMBC图,证实了上述分析,而且进一步说明,羰基取代在2为上。
综合上述分析可推知其为:对映-贝壳杉-16α,17-二羟基-2酮[ent-kauran(5R,8S,9R,10R,13R,16R)-2-one-16α,17-diol]。结构为下图:
化合物结构 化合物HMBC图
具体实施方式
本发明所述的新化合物是从山莓叶经过反复硅胶柱层析、薄层层析和结晶等方法分离出的,化学名为:对映-贝壳杉-16α,17-二羟基-2酮[ent-kauran(5R,8S,9R,10R,13R,16R)-2-one-16α,17-diol]。化合物的结构式为:
本发明提供的化合物的制备方法,包括以下步聚:
(1)提取:称取粉碎的山莓叶3500.0g,加入10倍体积80%的乙醇,搅匀,先超声提取30min,然后于室温下浸提48h,过滤,滤渣再重复提取一次,合并滤液,浓缩冻干得精粗提物,备用。
(2)液液萃取分离:将浸膏液依次用石油醚,氯仿、乙酸乙酯、正丁醇反复多次萃取,将萃取液分别浓缩,冷冻干。分别得石油醚萃取,氯仿、乙酸乙酯、正丁醇萃取物。
(3)柱层析及结晶分离纯化:将乙酸乙酯萃取物进行硅胶柱色谱分离,用正己烷与乙酸乙酯和乙酸乙酯与甲醇进行梯度洗脱,正己烷与乙酸乙酯的体积比依次为:100∶0,100∶50,乙酸乙酯与甲醇的体积比依次为100∶0,80∶1,40∶1,10∶1,5∶1,1∶1,0∶1。每瓶收集50mL,共收集173瓶,经TLC检测,显色,合并相同组份,共得15个组份,分别表示为:Fr.1,Fr.2,Fr.3,Fr.4,Fr.5,Fr.6,Fr.7,Fr.8,Fr.9,Fr.10,Fr.11,Fr.12,Fr.13,Fr.14Fr.15,Fr.2组份,经重结晶后得化合物100mg。
本发明中化合物抗肿瘤活性测定:
1、实验材料
1.1药品:上述分离的化合物:对映-贝壳杉-16α,17-二羟基-2酮。
1.2细胞:人肝癌细胞株HepG2由广州泰禾生物医药公司提供。
1.3试剂:PRMI1640,胎牛血清,96孔板,胰酶,DMSO,MTT,5-FU,青霉素,链霉素。
2、方法
将肝癌HepG2细胞培养于含10%胎牛血清、1x105U/L青霉素、链霉素的RPMI1640培养液中,37℃,5%CO2培养箱内常规传代培养。细胞以1x104个/孔的浓度接种于96孔培养板,于含10%的胎牛血清的RPMI-1640培养液中培养24小时,细胞贴壁后,实验组(化合物:对映-贝壳杉-16α,17二羟基-2酮)给药量为100μg/mL、200μg/mL、400μg/mL,阴性对照组加等量RPMI(DMSO含量为1%),阳性对照组加入5-Fu的浓度为100μg/mL、200μg/mL、400μg/mL、800μg/mL、1600μg/mL。空白对照组只加等量RPMI。药物作用24小时后,于每孔中加入配制的5%MTT 20μL继续培养4h,弃上清,加入150μL DMSO,在混合振荡仪上振药约10min,使结晶物充分溶解后,于酶联检测仪上492nm波长测定各孔吸光值。
计算细胞毒性值(CT%),CT%=(1一OD处理/OD对照)×100%。
3、实验结果(见下表)
化合物对肝癌HepG2细胞的抑制作用
上述实验结果表明:对映-贝壳杉-16α,17-二羟基-2酮化合物对肝癌HepG2细胞明显的抑制作用,且具有良好的量效关系。
Claims (5)
2.根据权利要求1所述的从山莓叶中分离出的化合物的制备方法,其特征在于,包括以下步骤:
(1)称取粉碎的山莓叶,加入10倍体积80%的乙醇,搅匀,先超声提取30min,然后于室温下浸提48h,过滤,滤渣再重复提取一次,合并滤液,浓缩冻干得精粗提物浸膏液,备用 ;
(2)将精粗提物浸膏液依次用石油醚、氯仿、乙酸乙酯、正丁醇反复多次萃取,将萃取液浓缩,冷冻干,收集乙酸乙酯萃取物 ;
(3)将乙酸乙酯萃取物进行硅胶柱色谱分离,用正己烷与乙酸乙酯和乙酸乙酯与甲醇进行梯度洗脱,合并流出液,经TLC检测,显色,合并相同组份,经重结晶后得该化合物。
3.根据权利要求2所述的从山莓叶中分离出的化合物的制备方法,其特征在于,进行梯度洗脱时,正己烷与乙酸乙酯的体积比依次为:100∶0,100∶50,乙酸乙酯与甲醇的体积比依次为100∶0,80∶1,40∶1,10∶1,5∶1,1∶1,0∶1。
4.根据权利要求1所述的从山莓叶中分离出的化合物作为制备抗肿瘤药物的用途。
5.根据权利要求1所述的从山莓叶中分离出的化合物作为制备抗肿瘤药物组分之一的用途。
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