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桔梗化学成分的提取分离
1 桔梗化学成分提取分离流程桔梗干燥根药材(10 kg),置于大型中药提取器中,以6倍量75%的乙醇浸泡过夜,加热回流提取3次,每次3 h,合并三次提取液,减压回收乙醇。将浓缩液用等体积的石油醚、乙酸乙酯分别萃取3次,剩余水溶液浓缩约3 L,经D101大孔吸附树脂,分别用水,30%乙醇水,60%乙醇水和95%乙醇水依次洗脱,得60%乙醇水洗脱部分浓缩后得浸膏90 g。60 %乙醇洗脱部分经(200-300目)柱色谱,以二氯甲烷-甲醇梯度洗脱(50:1;25:1;12:1;6:1;3:1;1:1)。将二氯甲烷-甲醇(1:1)馏分再经硅胶柱层析、反相ODS柱色谱,反相半制备高效液相色谱最终分离得到白色粉末状Compound 1,Compound 2;将二氯甲烷-甲醇(3:1)馏分再经硅胶柱层析、反相制备高效液相色谱,反相半制备高效液相色谱最终分离得到白色粉末状Compound 3和Compound 4。其具体分离流程如图1所示:图1 提取分离流程图
[img=,8,]file:///C:/Users/zhang/AppData/Local/Temp/ksohtml9420/wps11.png[/img][img=,8,]file:///C:/Users/zhang/AppData/Local/Temp/ksohtml9420/wps12.png[/img][img=,8,]file:///C:/Users/zhang/AppData/Local/Temp/ksohtml9420/wps13.png[/img][img=,8,]file:///C:/Users/zhang/AppData/Local/Temp/ksohtml9420/wps14.png[/img]2 化合物结构鉴定Compound 1:白色粉末(甲醇)。1H-NMR(300MHz ,pyridine-d5) δ:1.00(3H,s,H-29), 1.08(3H,s,H-30), 1.12(3H,s,H-26), 1.39(3H,s,H-27), 1.68(3H,s,H-25),1.68(3H,br.s,CH3-Rha)及不饱和双键氢δ5.63(1H,br.s,12-H)。13C-NMR(150MHz, pyridine-d5)δ:45.3(C-1), 68.7(C-2), 88.7(C-3), 48.2(C-4), 47.6(C-5), 19.4(C-6), 33.3(C-7), 40.6(C-8), 45.3(C-9), 38.0(C-10), 24.1(C-11), 123.3(C-12), 144.4(C-13), 42.5(C-14), 36.1(C-15), 74.0(C-16), 49.7(C-17), 41.6(C-18), 47.2(C-19), 31.0(C-20), 36.1(C-21), 32.2(C-22), 63.2(C-23), 67.5(C-24), 19.1(C-25), 17.7(C-26), 27.1(C-27), 176.0(C-28), 33.6(C-29), 24.8(C-30)。13C-NMR data of sugars:Glucose(inner): 106.1(C-1), 74.9(C-2), 78.4(C-3),72.4(C-4),76.5(C-5),70.4(C-6); Glucose(center): 105.0(C-1), 75.3(C-2),78.5(C-3), 71.3(C-4), 77.2(C-5),70.2(C-6); Glucose(terminal):105.6(C-1), 75.4(C-2),78.7(C-3),70.9(C-4),77.7(C-5),62.7(C-6); Arabinose: 93.6(C-1), 75.3(C-2), 71.6(C-3), 66.3(C-4), 63.3(C-5); Rhamnose: 101.2(C-1), 72.0(C-2), 72.7(C-3), 83.6(C-4), 68.7(C-5), 18.4(C-6); Xylose: 106.9(C-1), 76.1(C-2), 78.6(C-3), 71.1(C-4), 67.5(C-5)。该化合物数据与文献报道基本一致]。故鉴定化合物1为去芹糖桔梗皂苷E,结构式如图2所示。
图2 化合物1结构式
Compound 2:白色粉末(甲醇)。1H-NMR(300MHz,pyridine-d5)δ:1.00(3H,s,H-29), 1.11(3H,s,H-30), 1.08(3H,s,H-26), 1.36(3H,s,H-27), 1.69(3H,s,H-25),1.69(3H,br.s,CH3-Rha)及不饱和双键氢δ5.80(1H,br.s,12-H)。13C-NMR(150MHz ,pyridine-d5)δ:45.3(C-1),68.8(C-2),88.4(C-3),48.2(C-4),47.6(C-5),19.5(C-6),33.5(C-7),40.6(C-8),45.0(C-9),38.0(C-10),24.1(C-11),123.3(C-12),144.4(C-13),42.5(C-14),36.1(C-15),73.9(C-16),49.8(C-17),41.7(C-18),47.2(C-19),31.0(C-20),36.1(C-21),32.2(C-22),63.6(C-23),67.0(C-24),19.2(C-25),17.7(C-26),27.1(C-27),176.1(C-28),33.3(C-29),24.8(C-30)。13C-NMR data of sugars:Glucose(inner):106.1(C-1),74.9(C-2),78.4(C-3),72.4(C-4),76.6(C-5),70.6(C-6); Glucose(center): 105.0(C-1), 75.3(C-2),78.5(C-3), 71.3(C-4), 77.2(C-5),70.2(C-6); Glucose(terminal):105.7(C-1), 75.2(C-2),78.7(C-3),70.9(C-4),77.7(C-5),62.7(C-6); Arabinose: 93.7(C-1), 75.5(C-2), 71.6(C-3), 66.5(C-4), 63.1(C-5); Rhamnose: 101.3(C-1), 71.9(C-2), 72.8(C-3), 83.9(C-4), 68.6(C-5), 18.4(C-6); Xylose: 106.8(C-1), 75.8(C-2), 84.9(C-3), 69.5(C-4), 67.0(C-5); Apiose:111.3(C-1), 77.9(C-2), 80.6(C-3), 75.3(C-4), 65.4(C-5)。该化合物数据与文献报道基本一致,故鉴定化合物2为桔梗皂苷E,结构式如图3所示。
图3 化合物2结构式
Compound 3:白色粉末(甲醇)。1H-NMR(300MHz,pyridine-d5)δ:0.99(3H,s,H-29), 1.07(3H,s,H-30), 1.11(3H,s,H-26), 1.35(3H,s,H-27), 1.68(3H,s,H-25),1.68(3H,br.s,CH3-Rha)及不饱和双键氢δ5.63(1H,br.s,12-H)。13C-NMR(75MHz,pyridine-d5) δ:45.0(C-1), 68.2(C-2), 88.1(C-3), 47.2(C-4), 47.9(C-5), 19.2(C-6), 33.2(C-7), 40.2(C-8), 44.5(C-9), 37.7(C-10), 24.1(C-11), 123.3(C-12), 144.0(C-13), 42.1(C-14), 35.7(C-15), 73.6(C-16), 49.4(C-17), 41.3(C-18), 46.8(C-19), 30.6(C-20), 35.7(C-21), 31.8(C-22), 63.3(C-23), 67.1(C-24), 18.8(C-25), 17.3(C-26), 26.7(C-27), 175.7(C-28), 32.9(C-29), 24.4(C-30)。13C-NMR data of sugars:Glucose(inner):105.8(C-1),74.5(C-2),78.3(C-3),71.8(C-4),76.2(C-5),70.2(C-6); Glucose(terminal): 104.7(C-1),75.2(C-2),78.5(C-3),69.1(C-4),77.5(C-5),62.3(C-6); Arabinose: 93.3(C-1), 76.2(C-2), 66.2(C-3), 70.2(C-4), 62.7(C-5);Rhamnose: 100.9(C-1), 71.1(C-2), 72.4(C-3), 83.6(C-4), 68.4(C-5), 18.1(C-6);Xylose: 106.4(C-1), 75.2(C-2), 84.4(C-3), 70.2(C-4), 66.6(C-5); Apiose:110.9(C-1), 77.5(C-2), 80.2(C-3), 74.9(C-4), 65.0(C-5)。该化合物数据与文献报道基本一致[。故鉴定化合物3为桔梗皂苷D3,结构式如图4所示。
图4 化合物3结构式
Compound 4:白色粉末(甲醇)。1H-NMR(600MHz,pyridine-d5)δ:0.98(3H,s,H-29), 1.13(3H,s,H-26), 1.14(3H,s,H-30), 1.33(3H,s,H-25), 1.56(3H,s,H-24),1.76(3H,s,H-27), 1.70(3H,br.s,CH3-Rha)及不饱和双键氢δ5.72(1H,br.s, H-12)。13C-NMR(150MHz,pyridine-d5) δ:44.4(C-1), 69.7(C-2), 83.7(C-3), 42.9(C-4), 47.7(C-5), 18.1(C-6), 33.3(C-7), 40.2(C-8), 47.8(C-9), 37.0(C-10), 24.1(C-11), 123.2(C-12), 144.4(C-13), 42.3(C-14), 36.0(C-15), 74.1(C-16), 49.6(C-17), 41.3(C-18), 47.1(C-19), 32.2(C-20), 36.2(C-21), 31.0(C-22), 65.2(C-23), 15.1(C-24), 17.7(C-25), 17.4(C-26), 27.3(C-27), 176.0(C-28), 33.3(C-29), 24.8(C-30)。13C-NMR data of sugars:Glucose(inner):105.9(C-1),74.2(C-2),88.7(C-3),72.0(C-4),77.8(C-5),70.8(C-6); Glucose(terminal): 105.4(C-1),75.4(C-2),78.8(C-3),69.4(C-4),78.3(C-5),62.3(C-6); Arabinose: 93.5(C-1), 75.6(C-2), 66.0(C-3), 69.9(C-4), 62.6(C-5);Rhamnose: 101.2(C-1), 71.7(C-2), 72.7(C-3), 83.0(C-4), 68.6(C-5), 18.4(C-6);Xylose: 106.7(C-1), 75.2(C-2), 84.8(C-3), 69.1(C-4), 67.0(C-5); Apiose:111.3(C-1), 77.9(C-2), 80.5(C-3), 75.3(C-4), 65.4(C-5)。该化合物数据与文献报道基本一致。故鉴定化合物4为远志皂苷D2,结构式如图2所示。
图5 化合物4结构式
3 小结 采用75 %乙醇回流提取,再分别用石油醚、乙酸乙酯进行萃取,剩余水溶液浓缩后通过D101大孔吸附树脂,对60 %乙醇洗脱液进行提取分离。通过采用反复硅胶柱色谱法、ODS柱色谱和制备以及半制备高效液相色谱法等分离纯化手段,共分离纯化得到4个化合物,均为桔梗皂苷类化合物,鉴定为:去芹糖桔梗皂苷E(Deapio-platycoside E)、桔梗皂苷E(Platycoside E)、桔梗皂苷D3(Platycodin D3)和远志皂苷D2(Polygalacin D2)。 桔梗中主要药理活性成分为桔梗皂苷,含量较大,利用传统的萃取技术来纯化大量皂苷震摇剧烈容易产生乳化现象,不利于纯化,因此先使用大孔吸附树脂对皂苷成分进行前期纯化,有助于富集大量的皂苷进行后续的单体化合物的分离。 桔梗皂苷为齐墩果酸型五环三萜类皂苷,一般母核连有3~7个糖基,极性较大,而且结构相近较难分离,采用硅胶柱层析等正相分离手段无法较好的分离,必须较多的借助反复ODS柱色谱,制备高效液相色谱等反相分离技术得以分离得到较纯的单体化合物。