苦地丁中生物碱成分研究

苦地丁中生物碱成分研究

苦丁是紫堇科植物的一种干燥带，具有清热解毒、活血消肿的功效，对各种炎症（如感冒、发烧、肝炎等）也有良好疗效。苦地丁总生物碱有镇静催眠和抗惊厥作用,其主要成分紫堇灵、乙酰紫堇灵、原阿片碱对肝损伤有保护作用。到目前为止已从苦地丁中分得生物碱近二十个,其类型主要为苯菲啶型和原小檗碱型异喹啉生物碱,本研究从该植物中分离并鉴定出16种生物碱,其中coryincine、紫堇萨明(corysamine)、去氢碎叶紫堇碱(dehydrocheilanthifoline)、黄连碱(coptisine)、氧化血根碱(oxysanguinarine)、去甲血根碱(norsanguinarine)、6-丙酮基二氢血根碱(6-acetonyldihydrosanguinarine)、spallidamine为首次从苦地丁中分得;N-反式-阿魏酰基酪胺(N-trans-feruloyltyramine)为首次从紫堇属植物中分得。1质谱及定量分析熔点用X-4型显微熔点测定仪测定,温度未经校正;核磁共振用BrukerACF-300核磁共振仪测定;质谱用Agilent1100MSDTrap电喷雾质谱仪测定。柱层析及薄层层析用硅胶均为青岛海洋化工厂生产。其余所用试剂均为分析纯。苦地丁(河北玉田镇新兴产业园区),经作者鉴定为紫堇属植物苦地丁(CorydalisbungeanaTurcz.)。2反渗透膜流份的制备苦地丁干燥全草8kg,粉碎后用90%乙醇加热回流提取3次,减压回收乙醇,浸膏用2%盐酸研磨,用石油醚萃取3次,回收溶剂得石油醚浸膏320g;盐酸液用氨水调pH至10,用氯仿反复萃取,合并氯仿液并回收氯仿得总碱82g。总碱82g经硅胶柱层析分离,石油醚-氯仿、氯仿-甲醇梯度洗脱,得13个流份(A～M)。流份A以石油醚-乙酸乙酯为洗脱剂,硅胶柱层析分离得碱Ⅲ(85mg);流份C以环己烷-丙酮为洗脱剂,反复硅胶柱层析分离纯化,得碱Ⅰ(10g)、碱Ⅱ(20mg)、碱Ⅳ(6mg)、碱Ⅴ(6mg)、碱Ⅵ(30mg)、碱ⅩⅢ(120mg);流份L以氯仿-甲醇为洗脱剂,反复硅胶柱层析,再经SephadexLH-20凝胶柱层析得碱Ⅻ(5mg);其余流份以氯仿-甲醇为洗脱剂,反复硅胶柱层析得碱Ⅶ(30mg)、碱Ⅷ(130mg)、碱Ⅸ(720mg)、碱Ⅹ(50mg)、碱Ⅺ(20mg)。取石油醚浸膏260g,以石油醚-乙酸乙酯为洗脱剂,反复硅胶柱层析分离纯化,得碱ⅩⅣ(25mg)、碱ⅩⅤ(4mg)和碱ⅩⅥ(6mg)。3碱为n-反式-阿魏酰基酪胺cd3,a碱Ⅰ、Ⅲ～Ⅶ、Ⅸ苦地丁中已分离到的生物碱,通过理化性质、ESI-MS、1HNMR、13CNMR的综合分析以及与文献对照,分别鉴定为:紫堇灵(corynoline)、二氢血根碱(dihydrosanguinarine)、(+)-13-表紫堇灵[(+)-13-epicorynoline]、四氢紫堇萨明(tetrahydrocorysamine)、四氢黄连碱(tetrahydrocoptisine)、6,7-methylenedioxy-2-(6-acetyl-2,3-methylenedioxybenzyl)-1(2H)-isoquinolinone、普托品(protopine)。碱Ⅱ无色针晶(甲醇),改良碘化铋钾试剂显色呈阳性。mp:210～211℃,分子式:C22H19NO6。ESI-MSm/z:394[M+H]+。1HNMR(CDCl3)δ:0.89(3H,s,13-CH3),1.87(3H,s,-COCH3),2.94(1H,d,J=18.6Hz,12-Hβ),3.25(1H,dd,J=18.6Hz,4.2Hz,12-Hα),4.69(1H,br.s,14-H),5.77(1H,d,J=4.2Hz,11-H),5.91,6.06(4H,2×-OCH2O-),6.59(1H,s,1-H),6.66(1H,d,J=8.1Hz,9-H),6.83(1H,d,J=8.1Hz,10-H),7.68(1H,s,4-H),8.71(1H,d,J=3.6Hz,6-H)。以上数据与coryincine数据基本一致,鉴定碱Ⅱ为coryincine。碱Ⅷ白色粉末,紫外灯下(365nm)显蓝色荧光。分子式:C18H19NO4。ESI-MSm/z:314[M+H]+,336[M+Na]+,352[M+K]+。1HNMR(CD3OD)δ:3.88(3H,s,-OCH3),6.40(1H,d,J=15.6Hz,7′-H),6.80(1H,d,J=8.1Hz,5′-H),7.02(1H,dd,J=8.1Hz,2.0Hz,6′-H),7.12(1H,d,J=2.0Hz,2′-H),7.43(1H,d,J=15.6Hz,8′-H),2.75(2H,t,J=7.5Hz,7-H),3.47(2H,t,J=7.5Hz,8-H),6.72(2H,dd,J=8.4Hz,2.1Hz,3,5-H),7.06(2H,dd,J=8.4Hz,2.1Hz,2,6-H)。13CNMR(CD3OD)δ:51.4(-OCH3),111.6(C-2′),116.5(C-5′),118.8(C-8′),123.2(C-6′),128.3(C-1′),142.0(C-7′),149.3(C-3′),149.9(C-4′),169.2(C=O),35.8(C-7),42.5(C-8),116.3(C-3,5),130.7(C-2,6),131.3(C-1),157.0(C-4)。以上数据与N-反式-阿魏酰基酪胺(N-trans-feruloyltyramine)数据基本一致,推定碱Ⅷ为N-反式-阿魏酰基酪胺。碱Ⅹ黄色粉末,易溶于甲醇,紫外灯下(365nm)显黄绿色荧光,改良碘化铋钾试剂显色呈阳性。分子式:C20H16NO4♁。ESI-MSm/z:334[M]+。1HNMR(CD3OD)δ:2.98(3H,s,13-CH3),3.16(2H,t,J=6.0Hz,5-H),4.77(2H,t,J=6.0Hz,6-H),6.12(2H,s,OCH2O-2,3),6.48(2H,s,OCH2O-9,10),7.03(1H,s,4-H),7.34(1H,s,1-H),7.91(1H,d,J=6.9Hz,12-H),8.00(1H,d,J=6.9Hz,11-H),9.71(1H,s,8-H)。13CNMR(CD3OD)δ:18.6(C13-CH3),29.1(C-5),58.6(C-6),103.6(OCH2O-2,3),106.3(OCH2O-9,10),109.2(C-1),111.8(C-4),112.9(C-11),120.6(C-12),121.4(C-1a),121.9(C-4a),132.8(C-13),134.5(C-12a),134.8(C-8a),137.6(C-14),143.8(C-8),146.7(C-10),148.6(C-9),149.2(C-2),151.3(C-3)。以上数据与紫堇萨明(corysamine)数据基本一致,故鉴定碱Ⅹ为紫堇萨明。碱Ⅺ棕红色针晶(甲醇),紫外灯下(365nm)显橙黄色荧光,改良碘化铋钾试剂显色呈阳性。分子式:C19H16NO4♁。ESI-MSm/z:322[M]+。1HNMR(CD3OD)δ:3.26(2H,t,J=6.4Hz,5-H),3.98(3H,s,-OCH3),4.89(2H,t,J=6.4Hz,6-H),6.46(2H,s,OCH2O-10,11),7.02(1H,s,4-H),7.55(1H,s,1-H),7.87(2H,s,11,12-H),8.68(1H,s,13-H),9.70(1H,s,8-H)。13CNMR(CD3OD)δ:27.8(C-5),56.7(C-6),57.5(-OCH3),106.1(OCH2O-9,10),112.1(C-1),113.1(C-4),113.7(C-1a),120.7(C-8a),122.1(C-11),122.2(C-12),123.0(C-13),128.4(C-4a),134.6(C-12a),139.3(C-14),145.2(C-8),145.7(C-10),148.3(C-9),149.1(C-3),152.5(C-2)。1HNMR数据与去氢碎叶紫堇碱(dehydrocheilanthifoline)数据一致,鉴定碱Ⅺ为去氢碎叶紫堇碱。碱Ⅻ黄色针晶(甲醇),紫外灯下(365nm)显黄色荧光,改良碘化铋钾试剂显色呈阳性。分子式:C19H14NO4♁。ESI-MSm/z:320[M]+。1HNMR(CD3OD)δ:3.26(2H,t,J=6.0Hz,5-H),4.89(2H,t,J=6.0Hz,6-H),6.11(2H,s,OCH2O-2,3),6.47(2H,s,OCH2O-9,10),6.96(1H,s,4-H),7.66(1H,s,1-H),7.87～7.89(2H,m,11,12-H),8.74(1H,s,13-H),9.71(1H,s,8-H)。1HNMR数据与黄连碱(coptisine)数据一致,鉴定碱Ⅻ为黄连碱。碱ⅩⅣ黄绿色粉末,紫外灯下(365nm)显淡黄色荧光,改良碘化铋钾试剂显色呈阳性。mp:277～279℃,分子式:C19H11NO4。ESI-MSm/z:318[M+H]+。1HNMR(CDCl3)δ:6.13(2H,s,OCH2O-2,3),6.29(2H,s,OCH2O-7,8),7.26(1H,s,1-H),7.46(1H,d,J=8.7Hz,9-H),8.16(1H,d,J=8.7Hz,10-H),7.84(1H,d,J=9.0Hz,12-H),8.31(1H,d,J=9.0Hz,11-H),8.70(1H,s,4-H),9.46(1H,s,6-H)。13CNMR(CDCl3)δ:101.4(OCH2O-2,3),102.3(C-4),102.7(OCH2O-7,8),104.4(C-1),112.8(C-6a),113.6(C-9),115.6(C-10),118.4(C-11),120.4(C-10b),127.4(C-12),128.2(C-10a),129.4(C-4a),129.8(C-12a),139.9(C-4b),143.6(C-7),144.8(C-6),145.2(C-8),148.4(C-2),148.6(C-3)。以上1HNMR、13CNMR数据与去甲血根碱(norsanguinarine)数据一致,故鉴定碱ⅩⅣ为去甲血根碱。碱ⅩⅤ无色针晶(甲醇),改良碘化铋钾试剂显色呈阳性。mp:191～192℃,分子式:C23H19NO5。ESI-MSm/z:390[M+H]+,346[M-COCH3]+,332[M-CH2COCH3]+。1HNMR(CDCl3)δ:2.08(3H,s,3′-CH3),2.35(1H,m,1′-H),2.70(3H,s,N-CH3),2.80(1H,m,1′-H),4.93(1H,dd,J=9.0Hz,5.0Hz,6-H),6.05(4H,s,2×-OCH2O-),6.87(1H,d,J=8.1Hz,9-H),7.11(1H,s,1-H),7.34(1H,d,J=8.1Hz,10-H),7.52(1H,d,J=8.6Hz,12-H),7.67(1H,s,4-H),7.70(1H,d,J=8.6Hz,11-H)。1HNMR数据与6-丙酮基二氢血根碱(6-acetonyldihydrosanguinarine)数据一致,推定碱ⅩⅤ为6-丙酮基二氢血根碱。碱ⅩⅥ灰白色粉末,改良碘化铋钾试剂显色呈阳性。分子式:C22H17NO6。ESI-MSm/z:392[M+H]+,332[M-CH2COOH]+。1HNMR(Acetone-d6)δ:2.35(1H,m,1′-H),2.68(3H,s,N-CH3),2.70(1H,m,1′-H),4.84(1H,q,J=4.3Hz,6-H),6.11(4H,s,2×-OCH2O-),6.92(1H,d,J=8.1Hz,9-H),7.20(1H,s,1-H),7.47(1H,d,J=8.1Hz,10-H),7.54(1H,s,4-H),7.57(1H,d,J=8.7Hz,12-H),7.80(1H,d,J=8.7Hz,11-H)。13CNMR(Acetone-d6)δ:39.6(C-1′),44.1(N-CH3),56.4(C-6),102.0(OCH2O-2,3),102.9(OCH2O-7,8),103.4(C-4),105.7(C-1),109