CN112608295B - 大叶补血草根酰胺木脂素类化合物及其制备方法和应用 - Google Patents
大叶补血草根酰胺木脂素类化合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种大叶补血草根中的酰胺木脂素类化合物及其制备方法和应用。该类化合物以大叶补血草为原料,用溶剂提取,溶剂萃取,通过硅胶柱层析法、聚酰胺柱层析法等方式进行分离,采取薄层层析法检测分析,得到8个新的酰胺木脂素类化合物和10个已知酰胺木脂素类化合物。通过降糖活性筛选发现所述新的酰胺木脂素类化合物及已知酰胺木脂素类化合物具有不同程度的降糖活性,可用于制备降糖药物中的应用,或将其与降糖药物组合制备降糖组合药物或保健品。
Description
技术领域
本发明为植物化学领域,具体为大叶补血草根酰胺木脂素类化合物及其制备方法和应用。
背景技术
大叶补血草(Limonium gmelinii),为白花丹科或蓝雪科(Plumbaginaceae)补血草属(Limonium Mill.)多年生草本盐生植物,分布较广,是补血草中群落面积最大的一种,国内主要分布在新疆北部,在伊犁地区、塔城地区和阿勒泰地区均有分布,为维吾尔族和哈萨克族的传统用药。国外对大叶补血草研究主要是哈萨克斯坦阿尔法拉比国立大学化学学院的G.E Zhusupova和L.M.Korulkina教授等人,从该植物的根部分离获得半乳糖、五倍子酸、丁香酸、鞣花酸和几种黄酮类化合物及其苷类。其中根部含多种黄酮醇。大叶补血草具有清热祛湿,止血散瘀,消炎,治疗子宫内膜炎、宫颈糜烂、子宫出血、尿血痈疽等功效,另外有记载了其民族民间的6种附方。大叶补血草在新疆地区有较丰富的植物资源,为了研究大叶补血草的化学成分并筛选具有疗效成分,本课题对大叶补血草进行了化学成分研究。查阅国内外文献,至今尚未见有关大叶补血草酰胺木脂素化学成分研究报道。
发明内容
本发明的目的在于,提供大叶补血草药材中的酰胺木脂素类化合物及其制备方法和应用。该类化合物以大叶补血草根药材为原料,用溶剂提取,溶剂萃取,通过硅胶柱层析法、反相柱层析法或葡聚糖凝胶LH-20柱层析法的三种或四种方式进行分离,采取薄层层析法检测分析,得到8个新的酰胺木脂素类化合物和10个已知酰胺木脂素类化合物,并对分离得到的8个新的酰胺木脂素类化合物及10个已知酰胺木脂素类化合物进行降糖活性筛选。结构表明:测试的酰胺木脂素中,化合物1-8、10-11和13-17对蛋白酪氨酸磷酸酶1B具有显著抑制作用,化合物1-6、8、10-11和13-15和17对α-葡萄糖苷酶具有显著抑制作用,抑制α-葡萄糖苷酶活性强于阳性对照阿卡波糖(IC50=1026.0±31.05μM)。
本发明所述的一种大叶补血草根中的酰胺木脂素类化合物,该类化合物的结构式为:
其中:化合物1的化学名称为5,10,11-三羟基-N-(4-羟苯乙基)-3-酮-3H-萘并[1,2-f]苯并呋喃-7-甲酰胺;
化合物2的化学名称为6,7-二羟苯基-1-(3-甲氧基-4-羟苯基)-N-[2-(4-羟苯基)乙基]-苯并[f]异吲哚-2,3-二酮;
化合物3的化学名称为4-(3,4-二羟苯基)-6-羟基-2-(4-羟基苯乙基)-7-甲氧基-1H-苯并[f]异吲哚-1,3(2H)-二酮;
化合物4的化学名称为4-(3,4-二羟苯基)-6-羟基-2-(4-羟基-3-甲氧苯乙基)-7-甲氧基-1H-苯并[f]异吲哚-1,3(2H)-二酮;
化合物5的化学名称为6-羟基-2-(4-羟基苯乙基)-7-甲氧基-1H-苯并[f]异吲哚-1,3(2H)-二酮;
化合物6的化学名称为(2,3-顺)-2-(3,4-二氢苯基)-4-((Z)-4-羟基-3-甲氧苯基)-N,1-双(4-羟基苯乙基)-5-酮代吡咯烷-3-甲酰胺;
化合物7的化学名称为(1,2-顺)-6,7-二羟基-1-(4-羟基-3-甲氧苯基)-N2,N3-双-(4-对羟基苯乙酸)-乙基]-1,2-二氢萘-2,3-二甲酰胺;
化合物8的化学名称为(1,2-反)-7,8二羟基-1-(4-羟基-3-甲氧苯基)-N2,N3-双(对羟基苯乙酸)-1,2-二氢萘-2,3-二甲酰胺;
其中化合物1为丁二酰亚胺并香豆素,化合物2-4为丁二酰亚胺类酰胺木脂素,化合物5为萘酰胺类木脂素,化合物6为内酰胺类酰胺木脂素,化合物7-8为芳基二氢萘类酰胺木脂素。
所述大叶补血草根中的酰胺木脂素类化合物的制备方法,按下列步骤进行:
a、取干燥的大叶补血草根粉碎,按料液比1:3-10,用浓度50-95%乙醇或无水甲醇室温下渗漏、冷浸、热回馏或超声提取1-3次,每次2-10小时,再将提取液用旋转蒸发仪减压蒸干,得到提取物;
b、将步骤a所得提取物用水分散,依次采用石油醚,乙酸乙酯和正丁醇进行萃取,通过旋转蒸发仪在温度40℃减压浓缩后得到各极性段的萃取液,然后浓缩至膏状,分别得到石油醚部位、乙酸乙酯部位和正丁醇部位;
c、将步骤b所得的乙酸乙酯部位用正相硅胶柱层析分离,用体积比100:0-1:1的氯仿-甲醇进行梯度洗脱,得到15个流分Fr.1-Fr.15;
d、将步骤c得到的流分Fr.2用反相色谱柱,体积比30:70-100:0的甲醇-水溶剂分离,得到7个小部位2C1-2C7,将小部位2C4用半制备高效液相色谱仪,体积比44:56的乙腈-水进行分离得到化合物5;
e、将步骤c得到的Fr.3用130×3cm的交联葡聚糖LH-20,溶剂无水甲醇进行分离,得到小部位3C1-3C6,将小部位3C4用半制备高效液相色谱仪体积比42:58的乙腈-水进行分离,得到化合物4;
f、将步骤c得到的Fr.4用40×5cm反相色谱柱,体积比20:80-100:0的甲醇-水进行分离,得到小部位4C1-4C8,将小部位4C3进行半制备高效液相色谱仪,体积比40:60的乙腈-水分离得到化合物16,将小部位4C5用半制备高效液相色谱仪体积比40:60的乙腈-水进行分离,得到化合物3,再将小部位4C6用半制备高效液相色谱仪体积比40:60的乙腈-水进行分离,得到化合物2;
g、将步骤c得到的Fr.5用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位5C1-5C7,将小部位5C3用半制备高效液相色谱仪体积比30:70乙腈-水进行分离,得到化合物7和8;
h、将步骤c得到的Fr.6用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位6C1-6C15,将小部位6C4用半制备高效液相色谱仪体积比35:65的乙腈-水进行分离,得到化合物6,再将小部位6C6用半制备高效液相色谱仪体积比30:70的乙腈-水进行分离,得到化合物1。
所述大叶补血草根中的酰胺木脂素类化合物在制备治疗降糖药物中的用途。
所述大叶补血草根中的酰胺木脂素类化合物在制备治疗降糖药物中的组合物的用途。
所述大叶补血草根中的酰胺木脂素类化合物在制备降糖保健品中的用途
所述大叶补血草根中的酰胺木脂素类化合物在制备降糖保健品添加剂的用途。
本发明所述的一种大叶补血草根中的酰胺木脂素类化合物,该类化合物中除了8个新化合物外还分离得到了10个已知化合物为:
化合物9为Oleraisoindole A;
化合物10为Cannabisin I;
化合物11为3,3′-demethyl–heliotropamide;
化合物12为Cannabisin D;
化合物13为Cannabisin B;
化合物14为Cannabisin C;
化合物15为Cannabisin A;
化合物16为(2,3-反式)-3-(3-羟基-5-甲氧苯基)-N-(4-羟基苯乙醇)-7-{(E)-3-[(4-羟基苯乙醇)氨基]-3-氧化丙烯-1-乙二胺-1-醇}-2,3-二氢苯并[b][1,4]二氧六环-2-甲酰胺;
化合物17为Cannabisin F;
化合物18为Thoreliamide B;
所述大叶补血草根中的酰胺木脂素类化合物的制备方法,按下列步骤进行:
a、取干燥的大叶补血草根粉碎,按料液比为1:3-10,用浓度50-95%乙醇或无水甲醇室温下渗漏、冷浸,热回馏、超声提取1-3次,每次2-10小时,将提取液用旋转蒸发仪减压蒸干,得到提取物;
b、将步骤a所得提取物用水分散,依次采用石油醚,乙酸乙酯和正丁醇进行萃取,通过旋转蒸发仪减压浓缩后得到各极性段的萃取液,然后浓缩至膏状,分别得到石油醚部位、乙酸乙酯部位和正丁醇部位;
c、将步骤b所得的乙酸乙酯部位用正相硅胶柱层析分离,用体积比100:0-1:1的氯仿-甲醇进行梯度洗脱,得到15个流分Fr.1-Fr.15;
d、将Fr.2用反相色谱柱(LiChroprep RP-18),体积比30:70-100:0的甲醇-水分离,得到7个小部位2C1-2C7,将对小部位2C4用半制备高效液相色谱仪,体积比44:56乙腈-水进行分离得到化合物5,再将小部位2C5用半制备高效液相色谱仪体积比43:57乙腈-水进行分离,得到化合物9;
e、将步骤c得到的Fr.3用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位3C1-3C6,小部位3C4用半制备高效液相色谱仪,体积比42:58的乙腈-水进行分离,得到化合物4,将小部位3C3用半制备高效液相色谱仪,体积比42:58的乙腈-水进行分离,得到化合物17和18;
f、将步骤c得到的Fr.4用40×5cm反相色谱柱,体积比20:80-100:0的甲醇-水进行分离,得到小部位4C1-3C8,将小部位4C3进行半制备高效液相色谱仪,体积比40:60的乙腈-水分离得到化合物16,将小部位4C5用半制备高效液相色谱仪体积比40:60的乙腈-水进行分离,得到化合物3,再将小部位4C6和4C7用半制备高效液相色谱仪,体积比40:60的乙腈-水进行分离,得到化合物2和12;
g、将步骤c得到的Fr.5用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位5C1-5C7,将小部位5C3用半制备高效液相色谱仪体积比30:70的乙腈-水进行分离,得到化合物7,化合物8及化合物14;
h、将步骤c得到的Fr.6用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位6C1-6C15,将小部位6C4用半制备高效液相色谱仪,体积比35:65的乙腈-水进行分离,得到化合物6和11,再将小部位6C6用半制备高效液相色谱仪体积比30:70的乙腈-水进行分离,得到化合物1和10;
i、将步骤c得到的Fr.7用反相色谱柱(LiChroprep RP-18),体积比20:80-100:0的甲醇-水分离,得到小部位7C1-2C8,将小部位7C5用160×1.5cm交联葡聚糖LH-20,无水甲醇进行分离,得到化合物13和15。
本发明所述的大叶补血草根中的酰胺木脂素类化合物及其制备方法和用途,通过所述方法获得的8个新的和10已知酰胺木脂素类化合物经过降糖活性测定,实验结果表明:所述化合物1-18能够不同程度地抑制蛋白酪氨酸磷酸酶1B和α-葡萄糖苷酶的作用,从而表现出多降糖活性,可用于制备降糖药物或与降糖药物组合制备糖尿病组合药物和保健品。
本发明所述的大叶补血草根中的酰胺木脂素类化合物,可通过从植物中分离纯化得到,也可以经本领域技术人员熟知的化学修饰方法合成获得。
本发明所述的大叶补血草药材中的酰胺木脂素类化合物及其制备方法和应用。该类化合物采取薄层层析法检测分析,得到8个新的酰胺木脂素类化合物和10个已知酰胺木脂素类化合物,并对分离得到的8个新的酰胺木脂素类化合物及10个已知酰胺木脂素类化合物进行降糖活性筛选。结构表明:测试的酰胺木脂素中,化合物1-8、10-11和13-17对蛋白酪氨酸磷酸酶1B具有显著抑制作用,IC50值为1.71至11.27μM;化合物1-6、8、10-11和13-15和17对α-葡萄糖苷酶具有显著抑制作用,IC50值为1.50至29.16μM;抑制α-葡萄糖苷酶活性强于阳性对照阿卡波糖(IC50=1026.0±31.05μM);对所有化合物进行综合分析表明,化合物10(cannabisin I)和化合物17(cannabisin F)的抑制蛋白酪氨酸磷酸酶1B(cannabisinI的IC50值为2.01μM;cannabisin F的IC50值为1.71μM)和抑制α-葡萄糖苷酶(cannabisin I的IC50值为1.50μM;cannabisin F的IC50值为2.99μM)的活性最强,因此,cannabisin I和cannabisin F在糖尿病治疗中具有进一步开发利用价值;新化合物中Limoniumin B(化合物2)的降糖活性最强、即抑制蛋白酪氨酸磷酸酶1B的IC50为5.05μM,抑制α-葡萄糖苷酶的IC50为2.37μM。
本发明所述的大叶补血草根中的酰胺木脂素类化合物,采用高分辨质谱、一维和二维核磁共振谱等现代波谱手段确定其结构,结构鉴定过程如下:
化合物1,为黄色粉末状固体,薄层板喷浓硫酸-甲醇显色剂溶液,显黄色,HR-TOF-MS(m/z)给出的准分子离子峰[M-H]-456.10803(计算值C26H18O7,456.10888),确定分子式为C26H19NO7;
1H NMR和13C NMR数据见表1;
根据1H NMR和13C NMR及二维谱数据确定化合物1的结构为5,10,11-三羟基-N-(4-羟苯乙基)-3-酮-3H-萘并[1,2-f]苯并呋喃-7-甲酰胺;
化合物2,为黄色粉末状固体,薄层板喷浓硫酸-甲醇显色剂溶液,显黄色,HR-TOF-MS(m/z)给出的准分子离子峰[M-H]-470.12332(计算值C27H20NO7,470.12453),确定分子式为C27H21NO7;
1H NMR和13C NMR数据见表2;
根据1H NMR和13C NMR及二维谱数据确定化合物2的结构为6,7-二羟苯基-1-(3-甲氧基-4-羟苯基)-N-[2-(4-羟苯基)乙基]-苯并[f]异吲哚-2,3-二酮;
化合物3,为黄色粉末状固体,薄层板喷浓硫酸-甲醇显色剂溶液,显黄色,HR-TOF-MS(m/z)给出的准分子离子峰[M-H]-470.12485(计算值C27H20NO7,470.12453),确定分子式为C27H21NO7;
1H NMR和13C NMR数据见表2;
根据1H NMR和13C NMR及二维谱数据确定化合物3的结构为4-(3,4-二羟苯基)-6-羟基-2-(4-羟基苯乙基)-7-甲氧基-1H-苯并[f]异吲哚-1,3(2H)-二酮;
化合物4,为黄色粉末状固体,薄层板喷浓硫酸-甲醇显色剂溶液,显黄色,HR-TOF-MS(m/z)给出的准分子离子峰[M-H]-500.13434(计算值C28H22NO8,500.13509),确定分子式为C28H23NO8;
1H NMR和13C NMR数据见表2;
根据1H NMR和13C NMR及二维谱数据确定化合物4的结构为4-(3,4-二羟苯基)-6-羟基-2-(4-羟基-3-甲氧苯乙基)-7-甲氧基-1H-苯并[f]异吲哚-1,3(2H)-二酮;
化合物5,为黄色粉末状固体,薄层板喷浓硫酸-甲醇显色剂溶液,显黄色,HR-TOF-MS(m/z)给出的准分子离子峰[M-H]-362.10377(计算值C21H16NO5,362.10340),确定分子式为C21H17NO5;
1H NMR和13C NMR数据见表2;
根据1H NMR和13C NMR及二维谱数据确定化合物5的结构为6-羟基-2-(4-羟基苯乙基)-7-甲氧基-1H-苯并[f]异吲哚-1,3(2H)-二酮;
化合物6,为白色粉末状固体,HR-TOF-MS(m/z)给出的准分子离子峰[M-H]-609.22455(计算值C35H33N2O8,609.22424),确定分子式为C35H34N2O8;
1H NMR和13C NMR数据见表3;
根据1H NMR和13C NMR及二维谱数据确定化合物6的结构为(2,3-顺)-2-(3,4-二氢苯基)-4-((Z)-4-羟基-3-甲氧苯基)-N,1-双(4-羟基苯乙基)-5-酮代吡咯烷-3-甲酰胺;
化合物7,为白色粉末状固体,HR-TOF-MS(m/z)给出的准分子离子峰[M+H]+611.23520(计算值C35H35N2O8,611.23879),确定分子式为C35H34N2O8;
1H NMR和13C NMR数据见表3;
根据1H NMR和13C NMR及二维谱数据确定化合物8的结构为(1,2-顺)-6,7-二羟基-1-(4-羟基-3-甲氧苯基)-N2,N3-双-(4-对羟基苯乙酸)-乙基]-1,2-二氢萘-2,3-二甲酰胺;
化合物8,为白色粉末状固体,HR-TOF-MS(m/z)给出的准分子离子峰[M+H]+611.23578(计算值C35H35N2O8,611.23879),确定分子式为C35H34N2O8;
1H NMR和13C NMR数据见表3;
8个新化合物的结构鉴定:
表1.化合物11H and 13C核磁二甲亚砜数据(δin ppm,J in Hz)
表2.化合物2~5 1H and 13C核磁数据(δin ppm,J in Hz)
表3.化合物6~8 1H and 13C核磁数据(δin ppm,J in Hz)
具体实施方式
实施例1
a、取干燥的大叶补血草根20kg粉碎,按料液比为1:3,用无水甲醇室温下渗漏提取1次,时间5小时,将提取液用旋转蒸发仪减压蒸干,得到提取物;
b、将步骤a所得提取物用水分散,依次采用石油醚,乙酸乙酯和正丁醇进行萃取,通过旋转蒸发仪温度40℃减压浓缩后得到各极性段的萃取液,然后浓缩至膏状,分别得到石油醚部位、乙酸乙酯部位和正丁醇部位;
c、将步骤b所得的乙酸乙酯部位120g用正相硅胶柱层析分离,用体积比100:0-1:1的氯仿-甲醇进行梯度洗脱,得到15个流分Fr.1-Fr.15;
d、将步骤c所得的Fr.2用反相色谱柱(LiChroprep RP-18),体积比30:70-100:0的甲醇-水分离,得到7个小部位2C1-2C7,将小部位2C4用半制备高效液相色谱仪,体积比44:56乙腈-水进行分离得到化合物5(9mg),再将小部位2C5用半制备高效液相色谱仪体积比43:57乙腈-水进行分离,得到化合物9(7mg);
e、将步骤c得到的Fr.3(2.0g)用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位3C1-3C6,小部位3C4用半制备高效液相色谱仪,体积比42:58的乙腈-水进行分离,得到化合物4(4.5mg),将小部位3C3用半制备高效液相色谱仪,体积比42:58的乙腈-水进行分离,得到化合物化合物17(11mg)和化合物18(3.0mg);
f、将步骤c得到的Fr.4(4.5g)用40×5cm反相色谱柱,体积比20:80-100:0的甲醇-水进行分离,得到小部位4C1-3C8,将小部位4C3进行半制备高效液相色谱仪,体积比40:60的乙腈-水分离得到化合物16(3.5mg),将小部位4C5用半制备高效液相色谱仪体积比40:60的乙腈-水进行分离,得到化合物3(2.8mg),再将小部位4C6和4C7用半制备高效液相色谱仪,体积比40:60的乙腈-水进行分离,得到2化合物2(11mg)和化合物12(12mg);
g、将步骤c得到的Fr.5(3.3g)用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位5C1-5C7,将小部位5C3用半制备高效液相色谱仪体积比30:70的乙腈-水进行分离,得到化合物7(4.9mg),化合物8(3.5mg)及化合物14(3.9mg);
h、将步骤c得到的Fr.6(6.0g)用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位6C1-6C15,将小部位6C4用半制备高效液相色谱仪,体积比35:65的乙腈-水进行分离,得到化合物6(3.5mg)和化合物11(11mg),再将小部位6C6用半制备高效液相色谱仪体积比30:70的乙腈-水进行分离,得到化合物1(3.5mg)和化合物10(4.9mg)
i、将步骤c得到的Fr.7(2.0g)用反相色谱柱(LiChroprep RP-18),体积比20:80-100:0的甲醇-水分离,得到小部位7C1-2C8,将小部位7C5用160×1.5cm交联葡聚糖LH-20,无水甲醇进行分离,得到化合物13(4.0mg)和化合物15(4.5mg)。
实施例2
a、取干燥的大叶补血草根20kg粉碎,按料液比为1:5,用浓度70%乙醇室温下冷浸提取3次,每次2小时,将提取液用旋转蒸发仪减压蒸干,得到提取物;
b、将步骤a所得提取物用水分散,依次采用石油醚,乙酸乙酯和正丁醇进行萃取,通过旋转蒸发仪在温度40℃减压浓缩后得到各极性段的萃取液,然后浓缩至膏状,分别得到石油醚部位、乙酸乙酯部位和正丁醇部位;
c、将步骤b所得的乙酸乙酯部位取130g用正相硅胶柱层析分离,用体积比100:0-1:1的氯仿-甲醇进行梯度洗脱,得到15个流分Fr.1-Fr.15;
d、将Fr.2(4.0g)用反相色谱柱(LiChroprep RP-18),体积比30:70-100:0的甲醇-水分离,得到7个小部位2C1-2C7,将对小部位2C4用半制备高效液相色谱仪,体积比44:56乙腈-水进行分离得到化合物5(12mg),再将小部位2C5用半制备高效液相色谱仪体积比43:57乙腈-水进行分离,得到化合物9(9mg);
e、将步骤c得到的Fr.3(4.5g)用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位3C1-3C6,小部位3C4用半制备高效液相色谱仪,体积比42:58的乙腈-水进行分离,得到化合物4(6mg),将小部位3C3用半制备高效液相色谱仪,体积比42:58的乙腈-水进行分离,得到化合物17(11mg)和化合物18(2.8mg);
f、将步骤c得到的Fr.4(5.5g)用40×5cm反相色谱柱,体积比20:80-100:0的甲醇-水进行分离,得到小部位4C1-3C8,将小部位4C3进行半制备高效液相色谱仪,体积比40:60的乙腈-水分离得到化合物16(3.5mg),将小部位4C5用半制备高效液相色谱仪体积比40:60的乙腈-水进行分离,得到化合物3(3.3mg),再将小部位4C6和4C7用半制备高效液相色谱仪,体积比40:60的乙腈-水进行分离,得到化合物2(14mg)和化合物12(11mg);
g、将步骤c得到的Fr.5(3.8g)用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位5C1-5C7,将小部位5C3用半制备高效液相色谱仪体积比30:70的乙腈-水进行分离,得到化合物7(4.7mg),化合物8(2.9mg)及化合物14(3.8mg);
h、将步骤c得到的Fr.6(5.5g)用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位6C1-6C15,将小部位6C4用半制备高效液相色谱仪,体积比35:65的乙腈-水进行分离,得到化合物6(3.4mg)和化合物11(11mg),再将小部位6C6用半制备高效液相色谱仪体积比30:70的乙腈-水进行分离,得到化合物1(3.2mg)和化合物10(4.8mg);
i、将步骤c得到的Fr.7(3.4g)用反相色谱柱(LiChroprep RP-18),体积比20:80-100:0的甲醇-水分离,得到小部位7C1-2C8,将小部位7C5用160×1.5cm交联葡聚糖LH-20,无水甲醇进行分离,得到化合物13(3.4mg)和化合物15(4.7mg)。
实施例3
a、取干燥的大叶补血草根20kg粉碎,按料液比为1:7,用浓度80%乙醇室温下超声提取1次,时间10小时,将提取液用旋转蒸发仪减压蒸干,得到提取物;
b、将步骤a所得提取物用水分散,依次采用石油醚,乙酸乙酯和正丁醇进行萃取,通过旋转蒸发仪在温度40℃减压浓缩后得到各极性段的萃取液,然后浓缩至膏状,分别得到石油醚部位、乙酸乙酯部位和正丁醇部位;
c、将步骤b所得的乙酸乙酯部位用正相硅胶柱层析分离,用体积比100:0-1:1的氯仿-甲醇进行梯度洗脱,得到15个流分Fr.1-Fr.15;
d、将Fr.2(4.5g)用反相色谱柱(LiChroprep RP-18),体积比30:70-100:0的甲醇-水分离,得到7个小部位2C1-2C7,将对小部位2C4用半制备高效液相色谱仪,体积比44:56乙腈-水进行分离得到化合物5(13mg),再将小部位2C5用半制备高效液相色谱仪体积比43:57乙腈-水进行分离,得到化合物9(10mg);
e、将步骤c得到的Fr.3(4.2g)用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位3C1-3C6,小部位3C4用半制备高效液相色谱仪,体积比42:58的乙腈-水进行分离,得到化合物4(6mg),将小部位3C3用半制备高效液相色谱仪,体积比42:58的乙腈-水进行分离,得到化合物17(13mg)和化合物18(3.7mg);
f、将步骤c得到的Fr.4(5.5g)用40×5cm反相色谱柱,体积比20:80-100:0的甲醇-水进行分离,得到小部位4C1-3C8,将小部位4C3进行半制备高效液相色谱仪,体积比40:60的乙腈-水分离得到化合物16(3.5mg),将小部位4C5用半制备高效液相色谱仪体积比40:60的乙腈-水进行分离,得到化合物3(3.3mg),再将小部位4C6和4C7用半制备高效液相色谱仪,体积比40:60的乙腈-水进行分离,得到化合物2(13mg)和化合物12(11mg);
g、将步骤c得到的Fr.5(3.9g)用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位5C1-5C7,将小部位5C3用半制备高效液相色谱仪体积比30:70的乙腈-水进行分离,得到化合物7(4.9mg),化合物8(3.1mg)及化合物14(3.8mg);
h、将步骤c得到的Fr.6(7.0g)用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位6C1-6C15,将小部位6C4用半制备高效液相色谱仪,体积比35:65的乙腈-水进行分离,得到化合物6(3.8mg)和化合物11(1.9mg),再将小部位6C6用半制备高效液相色谱仪体积比30:70的乙腈-水进行分离,得到化合物1(3.5mg)和化合物10(4.8mg);
i、将步骤c得到的Fr.7(2.7g)用反相色谱柱(LiChroprep RP-18),体积比20:80-100:0的甲醇-水分离,得到小部位7C1-2C8,将小部位7C5用160×1.5cm交联葡聚糖LH-20,无水甲醇进行分离,得到化合物13(3.8mg)和化合物15(4.4mg)。
实施例4
a、取干燥的大叶补血草根20kg粉碎,按料液比为1:3-5,用浓度95%乙醇室温下热回馏提取2次,每次3小时,将提取液用旋转蒸发仪减压蒸干,得到提取物;
b、将步骤a所得提取物用水分散,依次采用石油醚,乙酸乙酯和正丁醇进行萃取,通过旋转蒸发仪在温度40℃减压浓缩后得到各极性段的萃取液,然后浓缩至膏状,分别得到石油醚部位、乙酸乙酯部位和正丁醇部位;
c、将步骤b所得的乙酸乙酯部位用正相硅胶柱层析分离,用体积比100:0-1:1的氯仿-甲醇进行梯度洗脱,得到15个流分Fr.1-Fr.15;
d、将Fr.2(3.9g)用反相色谱柱(LiChroprep RP-18),体积比30:70-100:0的甲醇-水分离,得到7个小部位2C1-2C7,将对小部位2C4用半制备高效液相色谱仪,体积比44:56乙腈-水进行分离得到化合物5(13mg),再将小部位2C5用半制备高效液相色谱仪体积比43:57乙腈-水进行分离,得到化合物9(10mg);
e、将步骤c得到的Fr.3(4.7g)用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位3C1-3C6,小部位3C4用半制备高效液相色谱仪,体积比42:58的乙腈-水进行分离,得到化合物4(7mg),将小部位3C3用半制备高效液相色谱仪,体积比42:58的乙腈-水进行分离,得到化合物17(14mg)和化合物18(3.7mg);
f、将步骤c得到的Fr.4(5.5g)用40×5cm反相色谱柱,体积比20:80-100:0的甲醇-水进行分离,得到小部位4C1-3C8,将小部位4C3进行半制备高效液相色谱仪,体积比40:60的乙腈-水分离得到化合物16(4.5mg),将小部位4C5用半制备高效液相色谱仪体积比40:60的乙腈-水进行分离,得到化合物3(4.5mg),再将小部位4C6和4C7用半制备高效液相色谱仪,体积比40:60的乙腈-水进行分离,得到化合物2(14.5mg)和12(12.3mg);
g、将步骤c得到的Fr.5(5.2g)用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位5C1-5C7,将小部位5C3用半制备高效液相色谱仪体积比30:70的乙腈-水进行分离,得到化合物7(6.5mg),化合物8(4.1mg)及化合物14(4.6mg);
h、将步骤c得到的Fr.6(7.2g)用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位6C1-6C15,将小部位6C4用半制备高效液相色谱仪,体积比35:65的乙腈-水进行分离,得到化合物6(5.3mg)和化合物11(12.1mg),再将小部位6C6用半制备高效液相色谱仪体积比30:70的乙腈-水进行分离,得到化合物1(5.3mg)和化合物10(5.5mg);
i、将步骤c得到的Fr.7(4.5g)用反相色谱柱(LiChroprep RP-18),体积比20:80-100:0的甲醇-水分离,得到小部位7C1-2C8,将小部位7C5用160×1.5cm交联葡聚糖LH-20,无水甲醇进行分离,得到化合物13(4.5mg)和化合物15(6.1mg)。
实施例5
化合物1-18对两种糖尿病相关蛋白酪氨酸磷酸酶1B和α-葡萄糖苷酶抑制作用测试:材料与试剂:实验仪器:Binder公司的细胞培养箱;美国Molecular Devices的SpectraMax MD5酶标仪;Leica公司的电子显微镜;Beckman Coulter公司的低温离心机;Eppendorf公司的微量移液器;Sartorius公司的PB-10型酸度计;Invitrogen公司的细胞计数仪;Sartorius公司的LA120S型电子天平;
实验耗材与试剂:Corning公司的96孔板培养瓶、细胞挂棒和细胞培养板;Amresco公司的DMSO;Oriental Yeast CO.,LTD公司的α-葡萄糖苷酶;Sigma-Aldrich公司的阿卡波糖;Multiple Science Biotech公司的竞争性ELISA试剂盒;GraphPad Software公司Graphpad Prism 5软件。
实验方法:
化合物1-18对蛋白酪氨酸磷酸酶1B抑制作用的测试:
以对-硝基苯基磷酸二钠(pNPP)作为底物,根据蛋白酪氨酸磷酸酶1B水解对-硝基苯基磷酸二钠的磷酸基团而产生颜色反应来测定蛋白酪氨酸磷酸酶1B的活性。缓冲液(20mM羟乙基哌嗪乙硫磺酸,150mM氯化钠,和1mM乙二胺四乙酸),在96孔板中加入含蛋白酪氨酸磷酸酶1B溶液的缓冲液179μL,测试样品或阳性对照样品1μL(将样品配成浓度梯度)或二甲亚砜1μL,混匀,10分钟后加入35mM的对-硝基苯基磷酸二钠20μL 25℃避光孵育30分钟后,每孔加10μL 3M氢氧化钠终止反应。以不含酶溶液的系统为空白,利用SpectraMax MD5酶标仪(美国Molecular Devices)在405nm测定吸收值。抑制率(I%)=[酶活组-(药品组-药品对照组)/(酶活组-酶活对照组)]×100%,IC50用软件计算。参照化合物为对酶蛋白酪氨酸磷酸酶1B抑制剂[3-(3,5-二溴-4-羟基苯甲酰丙烯酸)-2-乙基苯并呋喃-6-硫酸-[4-(噻唑-2-胺磺酰基)-苯基]酰胺];
大叶补血草中化合物1-18对α-葡萄糖苷酶抑制作用的测试:
实验以对硝基苯酚吡喃葡萄糖苷为底物,通过检测产物硝基苯酚吡喃葡萄糖苷的变化,确定α-葡萄糖苷酶的活性。反应总体积为100μL:2μL待测样品,71.5μL pH 6.8的磷酸盐缓冲溶液,1.5μLα-葡萄糖苷酶,室温孵育10分钟后加入25μL,10mmol/L硝基苯酚吡喃葡萄糖苷,振荡混匀,温度37℃反应30分钟后,405nm处测定吸收值,以不含酶溶液体系为空白,以阿卡波糖为阳性对照。待测样品:二甲基亚砜溶解待测样品。利用SpectraMax MD5酶标仪(美国Molecular Devices)在405nm测定吸收值;
抑制率(I%)=[酶活组-(药品组-药品对照组)/(酶活组-酶活对照组)]×100%,IC50用软件计算;
表4.大叶补血草根中化合物1-18对蛋白酪氨酸磷酸酶1B和α-葡萄糖苷酶抑制作用
a[3-(3,5-二溴-4-羟基苯甲酰丙烯酸)-2-乙基苯并呋喃-6-硫酸-[4-(噻唑-2-胺磺酰基)-苯基]酰胺]。
由表4可知,测试的酰胺木脂素中,新化合物1-8和已知化合物10-11、13-17对蛋白酪氨酸磷酸酶1B具有显著抑制作用,IC50值为1.71至11.27μM;新化合物1-8和已知化合物10-18对α-葡萄糖苷酶具有显著抑制作用,IC50值为1.50至29.16μM;即8个新化合物和10个已知化合物抑制α-葡萄糖苷酶活性强于阳性对照阿卡波糖(IC50=1026.0±31.05μM);对所有化合物进行综合分析表明,新化合物1-8具有较强降糖活性、即抑制蛋白酪氨酸磷酸酶1B的IC50为3.24至9.77μM,抑制α-葡萄糖苷酶的IC50为2.37至70.11μM。
Claims (6)
1.一种大叶补血草根中的酰胺木脂素类化合物,其特征在于该类化合物的结构式为:
其中:化合物1的化学名称为5,10,11-三羟基-N-(4-羟苯乙基)-3-酮-3H-萘并[1,2-f]苯并呋喃-7-甲酰胺;
化合物2的化学名称为6,7-二羟苯基-1-(3-甲氧基-4-羟苯基)-N-[2-(4-羟苯基)乙基]-苯并[f]异吲哚-2,3-二酮;
化合物3的化学名称为4-(3,4-二羟苯基)-6-羟基-2-(4-羟基苯乙基)-7-甲氧基-1H-苯并[f]异吲哚-1,3(2H)-二酮;
化合物4的化学名称为4-(3,4-二羟苯基)-6-羟基-2-(4-羟基-3-甲氧苯乙基)-7-甲氧基-1H-苯并[f]异吲哚-1,3(2H)-二酮;
化合物5的化学名称为6-羟基-2-(4-羟基苯乙基)-7-甲氧基-1H-苯并[f]异吲哚-1,3(2H)-二酮;
化合物6的化学名称为(2,3-顺)-2-(3,4-二氢苯基)-4-((Z)-4-羟基-3-甲氧苯基)-N,1-双(4-羟基苯乙基)-5-酮代吡咯烷-3-甲酰胺;
化合物7的化学名称为(1,2-顺)-6,7-二羟基-1-(4-羟基-3-甲氧苯基)-N2,N3-双-(4-对羟基苯乙酸)-乙基]-1,2-二氢萘-2,3-二甲酰胺;
化合物8的化学名称为(1,2-反)-7,8二羟基-1-(4-羟基-3-甲氧苯基)-N2,N3-双(对羟基苯乙酸)-1,2-二氢萘-2,3-二甲酰胺;
其中化合物1为丁二酰亚胺并香豆素,化合物2-4为丁二酰亚胺类酰胺木脂素,化合物5为萘酰胺类木脂素,化合物6为内酰胺类酰胺木脂素,化合物7-8为芳基二氢萘类酰胺木脂素。
2.根据权利要求1所述大叶补血草根中的酰胺木脂素类化合物的制备方法,其特征在于按下列步骤进行:
a、取干燥的大叶补血草根粉碎,按料液比1:3-10,用浓度50-95%乙醇或无水甲醇室温下渗漏、冷浸、热回馏或超声提取1-3次,每次2-10小时,再将提取液用旋转蒸发仪减压蒸干,得到提取物;
b、将步骤a所得提取物用水分散,依次采用石油醚,乙酸乙酯和正丁醇进行萃取,通过旋转蒸发仪在温度40℃减压浓缩后得到各极性段的萃取液,然后浓缩至膏状,分别得到石油醚部位、乙酸乙酯部位和正丁醇部位;
c、将步骤b所得的乙酸乙酯部位用正相硅胶柱层析分离,用体积比100:0-1:1的氯仿-甲醇进行梯度洗脱,得到15个流分Fr.1-Fr.15;
d、将步骤c得到的流分Fr.2用反相色谱柱,体积比30:70-100:0的甲醇-水溶剂分离,得到7个小部位2C1-2C7,将小部位2C4用半制备高效液相色谱仪,体积比44:56的乙腈-水进行分离得到化合物5;
e、将步骤c得到的Fr.3用130×3cm的交联葡聚糖LH-20,溶剂无水甲醇进行分离,得到小部位3C1-3C6,将小部位3C4用半制备高效液相色谱仪体积比42:58的乙腈-水进行分离,得到化合物4;
f、将步骤c得到的Fr.4用40×5cm反相色谱柱,体积比20:80-100:0的甲醇-水进行分离,得到小部位4C1-4C8,将小部位4C3进行半制备高效液相色谱仪,体积比40:60的乙腈-水分离得到化合物16,将小部位4C5用半制备高效液相色谱仪体积比40:60的乙腈-水进行分离,得到化合物3,再将小部位4C6用半制备高效液相色谱仪体积比40:60的乙腈-水进行分离,得到化合物2;
g、将步骤c得到的Fr.5用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位5C1-5C7,将小部位5C3用半制备高效液相色谱仪体积比30:70乙腈-水进行分离,得到化合物7和8;
h、将步骤c得到的Fr.6用130×3cm交联葡聚糖LH-20,无水甲醇进行分离,得到小部位6C1-6C15,将小部位6C4用半制备高效液相色谱仪体积比35:65的乙腈-水进行分离,得到化合物6,再将小部位6C6用半制备高效液相色谱仪体积比30:70的乙腈-水进行分离,得到化合物1。
3.根据权利要求1所述大叶补血草根中的酰胺木脂素类化合物在制备降糖药物中的用途。
4.根据权利要求1所述大叶补血草根中的酰胺木脂素类化合物在制备降糖药物组合物中的用途。
5.根据权利要求1所述大叶补血草根中的酰胺木脂素类化合物在制备降糖保健品中的用途。
6.根据权利要求1所述大叶补血草根中的酰胺木脂素类化合物在制备降糖保健品添加剂的用途。
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