苦地丁中生物堿成分研究

苦地丁中生物堿成分研究

苦丁是紫堇科植物的一種干燥帶，具有清熱解毒、活血消腫的功效，對(duì)各種炎癥（如感冒、發(fā)燒、肝炎等）也有良好療效?？嗟囟】偵飰A有鎮(zhèn)靜催眠和抗驚厥作用,其主要成分紫堇靈、乙酰紫堇靈、原阿片堿對(duì)肝損傷有保護(hù)作用。到目前為止已從苦地丁中分得生物堿近二十個(gè),其類型主要為苯菲啶型和原小檗堿型異喹啉生物堿,本研究從該植物中分離并鑒定出16種生物堿,其中coryincine、紫堇薩明(corysamine)、去氫碎葉紫堇堿(dehydrocheilanthifoline)、黃連堿(coptisine)、氧化血根堿(oxysanguinarine)、去甲血根堿(norsanguinarine)、6-丙酮基二氫血根堿(6-acetonyldihydrosanguinarine)、spallidamine為首次從苦地丁中分得;N-反式-阿魏酰基酪胺(N-trans-feruloyltyramine)為首次從紫堇屬植物中分得。1質(zhì)譜及定量分析熔點(diǎn)用X-4型顯微熔點(diǎn)測(cè)定儀測(cè)定,溫度未經(jīng)校正;核磁共振用BrukerACF-300核磁共振儀測(cè)定;質(zhì)譜用Agilent1100MSDTrap電噴霧質(zhì)譜儀測(cè)定。柱層析及薄層層析用硅膠均為青島海洋化工廠生產(chǎn)。其余所用試劑均為分析純?？嗟囟?河北玉田鎮(zhèn)新興產(chǎn)業(yè)園區(qū)),經(jīng)作者鑒定為紫堇屬植物苦地丁(CorydalisbungeanaTurcz.)。2反滲透膜流份的制備苦地丁干燥全草8kg,粉碎后用90%乙醇加熱回流提取3次,減壓回收乙醇,浸膏用2%鹽酸研磨,用石油醚萃取3次,回收溶劑得石油醚浸膏320g;鹽酸液用氨水調(diào)pH至10,用氯仿反復(fù)萃取,合并氯仿液并回收氯仿得總堿82g?？倝A82g經(jīng)硅膠柱層析分離,石油醚-氯仿、氯仿-甲醇梯度洗脫,得13個(gè)流份(A～M)。流份A以石油醚-乙酸乙酯為洗脫劑,硅膠柱層析分離得堿Ⅲ(85mg);流份C以環(huán)己烷-丙酮為洗脫劑,反復(fù)硅膠柱層析分離純化,得堿Ⅰ(10g)、堿Ⅱ(20mg)、堿Ⅳ(6mg)、堿Ⅴ(6mg)、堿Ⅵ(30mg)、堿ⅩⅢ(120mg);流份L以氯仿-甲醇為洗脫劑,反復(fù)硅膠柱層析,再經(jīng)SephadexLH-20凝膠柱層析得堿Ⅻ(5mg);其余流份以氯仿-甲醇為洗脫劑,反復(fù)硅膠柱層析得堿Ⅶ(30mg)、堿Ⅷ(130mg)、堿Ⅸ(720mg)、堿Ⅹ(50mg)、堿Ⅺ(20mg)。取石油醚浸膏260g,以石油醚-乙酸乙酯為洗脫劑,反復(fù)硅膠柱層析分離純化,得堿ⅩⅣ(25mg)、堿ⅩⅤ(4mg)和堿ⅩⅥ(6mg)。3堿為n-反式-阿魏?；野穋d3,a堿Ⅰ、Ⅲ～Ⅶ、Ⅸ苦地丁中已分離到的生物堿,通過(guò)理化性質(zhì)、ESI-MS、1HNMR、13CNMR的綜合分析以及與文獻(xiàn)對(duì)照,分別鑒定為:紫堇靈(corynoline)、二氫血根堿(dihydrosanguinarine)、(+)-13-表紫堇靈[(+)-13-epicorynoline]、四氫紫堇薩明(tetrahydrocorysamine)、四氫黃連堿(tetrahydrocoptisine)、6,7-methylenedioxy-2-(6-acetyl-2,3-methylenedioxybenzyl)-1(2H)-isoquinolinone、普托品(protopine)。堿Ⅱ無(wú)色針晶(甲醇),改良碘化鉍鉀試劑顯色呈陽(yáng)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)。以上數(shù)據(jù)與coryincine數(shù)據(jù)基本一致,鑒定堿Ⅱ?yàn)閏oryincine。堿Ⅷ白色粉末,紫外燈下(365nm)顯藍(lán)色熒光。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)。以上數(shù)據(jù)與N-反式-阿魏?；野?N-trans-feruloyltyramine)數(shù)據(jù)基本一致,推定堿Ⅷ為N-反式-阿魏?；野?。堿Ⅹ黃色粉末,易溶于甲醇,紫外燈下(365nm)顯黃綠色熒光,改良碘化鉍鉀試劑顯色呈陽(yáng)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)。以上數(shù)據(jù)與紫堇薩明(corysamine)數(shù)據(jù)基本一致,故鑒定堿Ⅹ為紫堇薩明。堿Ⅺ棕紅色針晶(甲醇),紫外燈下(365nm)顯橙黃色熒光,改良碘化鉍鉀試劑顯色呈陽(yáng)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數(shù)據(jù)與去氫碎葉紫堇堿(dehydrocheilanthifoline)數(shù)據(jù)一致,鑒定堿Ⅺ為去氫碎葉紫堇堿。堿Ⅻ黃色針晶(甲醇),紫外燈下(365nm)顯黃色熒光,改良碘化鉍鉀試劑顯色呈陽(yáng)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數(shù)據(jù)與黃連堿(coptisine)數(shù)據(jù)一致,鑒定堿Ⅻ為黃連堿。堿ⅩⅣ黃綠色粉末,紫外燈下(365nm)顯淡黃色熒光,改良碘化鉍鉀試劑顯色呈陽(yáng)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數(shù)據(jù)與去甲血根堿(norsanguinarine)數(shù)據(jù)一致,故鑒定堿ⅩⅣ為去甲血根堿。堿ⅩⅤ無(wú)色針晶(甲醇),改良碘化鉍鉀試劑顯色呈陽(yáng)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數(shù)據(jù)與6-丙酮基二氫血根堿(6-acetonyldihydrosanguinarine)數(shù)據(jù)一致,推定堿ⅩⅤ為6-丙酮基二氫血根堿。堿ⅩⅥ灰白色粉末,改良碘化鉍鉀試劑顯色呈陽(yáng)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