CN104231012B - 酚苷类化合物及其在制备抗补体药物中的用途 - Google Patents
酚苷类化合物及其在制备抗补体药物中的用途 Download PDFInfo
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Abstract
本发明属中药制药领域，涉及丹皮中酚苷类化合物及其在制备抗补体药物中的新用途。本发明采应用现代药理研究方法，对芍药属植物牡丹（Paeonia suffruticosa Andr.）的干燥根皮抗补体活性物质进行研究，从其95%乙醇提取物的正丁醇萃取部位分离得到8个酚苷类化合物，并证实其对补体系统经典途径和旁路途径均有较强的抑制作用。所述化合物对补体系统经典途径的抑制作用(CH50)为0.062‑0.185 mg/ml，对旁路途径的抑制作用(AP50)为0.102‑0.393mg/ml。所述化合物可用于制备补体抑制剂。
Description
技术领域
本发明属中药制药领域，涉及丹皮中酚苷类化合物及其在制备抗补体药物中的新用途。
背景技术
研究显示，补体系统的过度激活会引发系统性红斑狼疮、类风湿性关节炎、急性呼吸窘迫综合征等多种重大疾病。抗补体药物的研究多年来一直是世界药学研究的热点和重点，然而目前对上述因补体系统的过度激活引发的该类疾病尚缺乏较为理想的治疗药物，因此临床上急需高效、低毒、专一的新型补体抑制剂。从天然产物中研究开发补体抑制剂是近年来一个受到越来越多关注的重要研究领域，其具有成本低、毒性低等特点。国内外学者已从包括海洋生物在内的多种天然产物中分离得到多种具有补体系统抑制作用的单体化合物，为抗补体药物的研究与开发提供了广阔的前景。
丹皮始载于《神农本草经》，系毛茛科芍药属植物牡丹Paeonia suffruticosaAndr.的干燥根皮，味辛苦，性凉，归心、肝、肾经，具有清热凉血、活血化瘀之功效，用于温毒发斑、吐血钮血、夜热早凉、无汗骨蒸、经闭痛经、痈肿疮毒、跌扑伤痛。已有的有关丹皮化学成分研究表明，该植物主要含有单萜类、酚苷类、三萜类、黄酮类成分。近代药理研究表明，丹皮有抗凝血、降压、抗炎、抑制中枢神经系统等作用，但尚未见其抗补体活性的报道。
发明内容
本发明的目的是提供丹皮中具有抗补体活性的化合物，具体涉及其中8个酚苷类化合物：丹皮酚苷A(paeonolide A,1)、丹皮酚苷B(paeonolide B,2)、丹皮酚苷C(paeonolide C,3)、丹皮酚苷D(paeonolide D,4)、丹皮酚苷E(paeonolide E,5)、丹皮酚苷F(paeonolide F,6)、suffruticoside B(7)、suffruticoside D(8)。尤其是其中化合物1-6为新化合物。
本发明的进一步目的是提供上述丹皮中酚苷类化合物在制备抗补体药物中的用途。
本发明应用现代药理研究方法，对分离得到的单体化合物进行抗补体活性评价。从芍药属植物牡丹(Paeonia suffruticosa Andr.)的干燥根皮乙醇提取物的正丁醇萃取部位中分离得到8个酚苷类成分并证实其对补体系统经典途径和旁路途径均有较强的抑制作用，其中对补体系统经典途径的抑制作用(CH50)为0.062-0.185mg/ml，对旁路途径的抑制作用(AP50)为0.102-0.393mg/ml。本发明所述的酚苷类化合物可用于制备补体抑制剂，进一步制备抗补体药物。
本发明的活性酚苷类化合物具有以下结构通式的化学结构：
当R1＝R2＝G,R3＝H,R4＝AP,化合物为丹皮酚苷B(paeonolide B,1)；
当R1＝R2＝R3＝H,R4＝GAP,化合物为丹皮酚苷A(paeonolide A,2)；
当R1＝G,R2＝R3＝H,R4＝AP,化合物为丹皮酚苷C(paeonolide C,3)；
当R1＝R3＝H,R2＝G,R4＝AP,化合物为丹皮酚苷D(paeonolide D,4)；
当R1＝R3＝H,R2＝G,R4＝A,化合物为丹皮酚苷E(paeonolide E,5)；
当R1＝G,R2＝R3＝H,R4＝A,化合物为丹皮酚苷F(paeonolide F,6)；
当R1＝R2＝H,R3＝G,R4＝AP,化合物为suffruticoside B(7)；
当R1＝R2＝H,R3＝G,R4＝A,化合物为suffruticoside D(8)。
本发明所述的酚苷类化合物通过下述方法制备：
取丹皮20kg，粉碎，用95％乙醇室温冷浸(40L×3次)，合并提取液并浓缩得2.6kg的浸膏，取其中的1.0kg浸膏加水混悬，依次以等体积乙酸乙酯、正丁醇萃取(各2.0L×5次)，合并各萃取液并浓缩至干，得乙酸乙酯萃取物390g及正丁醇萃取物260g。取正丁醇部位浸膏加水混悬，上样于Diaion HP-20树脂，依次用水，25％、50％、75％、100％甲醇水溶液洗脱，得5个流份(PS-BU-1～5)。流份(PS-BU-2～4)分别经ODS柱色谱(甲醇：水，25:75-80:20)和Sephadex LH-20(甲醇)反复纯化，制备HPLC分离得到化合物丹皮酚苷A(paeonolideA,1)、丹皮酚苷B(paeonolide B,2)、丹皮酚苷C(paeonolide C,3)、丹皮酚苷D(paeonolideD,4)、丹皮酚苷E(paeonolide E,5)、丹皮酚苷F(paeonolide F,6)、suffruticoside B(7)、suffruticoside D(8)。
化合物1:丹皮酚苷A(paeonolide A)：黄白色固体；分子式：C34H36O20；分子量：764；1H-NMR(400MHz,CD3OD)δ:6.84(1H,s,H-3),6.65(1H,d,J＝8.6Hz,H-5),7.65(1H,d,J＝8.6Hz,H-6),2.37(3H,s,CH3),3.86(3H,s,OCH3),5.60(1H,d,J＝7.0Hz,H-1'),5.50(1H,m,H-2'),5.55(1H,m,H-3'),3.82(1H,m,H-4'),3.94(1H,m,H-5'),3.72(1H,d,J＝11.0,6.3Hz,H-6'),4.10(1H,d,J＝11.0Hz,H-6'),4.98(1H,b rs,H-1”),3.94(1H,m,H-2”),3.77(1H,d,J＝9.8Hz,H-4”),4.00(1H,d,J＝9.8Hz,H-4”),3.60(2H,s,H-5”),6.94(2H,s,H-2”',6”'),7.02(2H,s,H-2””,6””)；13C-NMR(100MHz,CD3OD)δ:122.8(C-1),159.5(C-2),102.9(C-3),166.2(C-4),109.2(C-5),133.1(C-6),32.3(CH3),200.3(C＝O),56.4(OCH3),99.8(C-1'),72.9(C-2'),76.9(C-3'),69.8(C-4'),77.3(C-5'),68.4(C-6'),111.1(C-1”),78.1(C-2”),80.6(C-3”),75.1(C-4”),65.6(C-5”),120.6(C-1”'),110.3(C-2”',6”'),146.4(C-3”',5”'),140.2(C-4”'),167.1(C-7”'),121.0(C-1””),110.4(C-2””,6””),146.3(C-3””,5””),140.0(C-4””),167.8(C-7””)。
化合物2:丹皮酚苷B(paeonolide B)：黄白色固体；分子式：C34H36O20；分子量：764；1H-NMR(400MHz,CD3OD)δ:6.75(1H,d,J＝2.3Hz,H-3),6.46(1H,dd,J＝8.6,2.3Hz,H-5),7.64(1H,d,J＝8.6Hz,H-6),2.61(3H,s,CH3),3.69(3H,s,OCH3),5.08(1H,d,J＝7.4Hz,H-1'),3.53(1H,m,H-2'),3.51(1H,m,H-3'),3.32(1H,m,H-4'),3.80(1H,m,H-5'),3.60(1H,m,H-6'),4.13(1H,d,J＝10.9Hz,H-6'),5.31(1H,d,J＝1.2Hz,H-1”),5.19(1H,J＝1.2Hz,H-2”),3.95(1H,d,J＝9.8Hz,H-4”),4.24(1H,d,J＝9.8Hz,H-4”),4.41(1H,J＝11.3Hz,H-5”),4.47(1H,J＝11.3Hz,H-5”),7.06(2H,s,H-2”',6”'),7.12(2H,s,H-2””,6””)；13C-NMR(100MHz,CD3OD)δ:122.3(C-1),160.3(C-2),103.2(C-3),166.2(C-4),108.4(C-5),133.0(C-6),32.1(CH3),200.7(C＝O),56.2(OCH3),102.0(C-1'),74.9(C-2'),78.3(C-3'),71.8(C-4'),77.0(C-5'),68.4(C-6'),108.8(C-1”),80.8(C-2”),79.9(C-3”),75.8(C-4”),68.0(C-5”),120.8(C-1”'),110.5(C-2”',6”'),146.5(C-3”',5”'),140.0(C-4”'),167.5(C-7”'),121.2(C-1””),110.3(C-2””,6””),146.4(C-3””,5””),139.9(C-4””),168.1(C-7””)。
化合物3:丹皮酚苷C(paeonolide C)：黄白色固体；分子式：C27H32O16；分子量：612；1H-NMR(400MHz,pyridine-d5)δ:7.23(1H,d,J＝2.3Hz,H-3),6.61(1H,dd,J＝9.0,2.3Hz,H-5),7.95(1H,d,J＝9.0Hz,H-6),2.81(3H,s,CH3),3.85(3H,s,OCH3),5.85(1H,d,J＝8.2Hz,H-1'),6.12(1H,t,J＝9.0Hz,H-2'),4.43(1H,t,J＝9.0Hz,H-3'),4.14(1H,m,H-4'),4.34(1H,m,H-5'),4.10(1H,dd,J＝10.2,6.6Hz,H-6'),4.78(1H,d,J＝10.2Hz,H-6'),5.69(1H,d,J＝2.4Hz,H-1”),4.74(1H,J＝2.4Hz,H-2”),4.37(1H,d,J＝9.0Hz,H-4”),4.63(1H,d,J＝9.0Hz,H-4”),4.18(2H,J＝5.0Hz,H-5”),7.88(2H,s,H-2”',6”')；13C-NMR(100MHz,pyridine-d5)δ:122.1(C-1),158.9(C-2),101.9(C-3),164.7(C-4),108.3(C-5),132.3(C-6),32.5(CH3),196.9(C＝O),55.8(OCH3),99.3(C-1'),74.5(C-2'),76.5(C-3'),71.6(C-4'),77.5(C-5'),68.9(C-6'),111.2(C-1”),77.7(C-2”),80.3(C-3”),75.0(C-4”),65.4(C-5”),121.0(C-1”'),110.4(C-2”',6”'),147.6(C-3”',5”'),141.2(C-4”'),166.7(C-7”')。
化合物4:丹皮酚苷D(paeonolide D)：黄白色固体；分子式：C27H32O16；分子量：612；1H-NMR(400MHz,CD3OD)δ:6.86(1H,d,J＝2.3Hz,H-3),6.69(1H,dd,J＝8.6,2.3Hz,H-5),7.76(1H,d,J＝8.6Hz,H-6),2.63(3H,s,CH3),3.88(3H,s,OCH3),5.18(1H,d,J＝7.8Hz,H-1'),3.79(1H,m,H-2'),5.26(1H,t,J＝9.0Hz,H-3'),3.68(1H,m,H-4'),3.82(1H,m,H-5'),3.63(1H,m,H-6'),4.05(1H,d,J＝10.2Hz,H-6'),4.96(1H,d,J＝2.7Hz,H-1”),3.91(1H,J＝2.3Hz,H-2”),3.75(1H,d,J＝9.8Hz,H-4”),3.97(1H,d,J＝9.8Hz,H-4”),3.58(2H,s,H-5”),7.15(2H,s,H-2”',6”')；13C-NMR(100MHz,CD3OD)δ:122.6(C-1),160.4(C-2),103.1(C-3),166.3(C-4),109.1(C-5),133.1(C-6),32.3(CH3),200.5(C＝O),56.3(OCH3),102.4(C-1'),77.0(C-2'),79.1(C-3'),69.8(C-4'),73.3(C-5'),68.5(C-6'),111.0(C-1”),78.0(C-2”),80.5(C-3”),75.0(C-4”),65.5(C-5”),121.6(C-1”'),110.4(C-2”',6”'),146.4(C-3”',5”'),139.8(C-4”'),168.1(C-7”')。
化合物5:丹皮酚苷E(paeonolide E)：黄白色固体；分子式：C27H32O16；分子量：612；1H-NMR(400MHz,pyridine-d5)δ:7.23(1H,d,J＝2.3Hz,H-3),6.64(1H,dd,J＝9.0,2.3Hz,H-5),7.98(1H,d,J＝9.0Hz,H-6),2.83(3H,s,CH3),3.88(3H,s,OCH3),5.89(1H,d,J＝8.2Hz,H-1'),6.08(1H,t,J＝9.4Hz,H-2'),4.44(1H,m H-3'),4.24(1H,m,H-4'),4.42(1H,m,H-5'),4.32(1H,dd,J＝10.5,6.6Hz,H-6'),4.83(1H,d,J＝10.5Hz,H-6'),4.84(1H,d,J＝7.0Hz,H-1”),4.41(1H,m,H-2”),4.13(1H,m,H-3”),4.29(1H,m,H-4”),3.70(1H,d,J＝10.6Hz,H-5”),4.30(1H,m,H-5”),7.89(2H,s,H-2”',6”')；13C-NMR(100MHz,pyridine-d5)δ:122.0(C-1),159.0(C-2),101.3(C-3),164.8(C-4),109.0(C-5),132.3(C-6),32.5(CH3),197.0(C＝O),56.0(OCH3),99.2(C-1'),74.5(C-2'),76.4(C-3'),71.5(C-4'),77.8(C-5'),70.0(C-6'),106.0(C-1”),72.3(C-2”),74.4(C-3”),69.3(C-4”),66.9(C-5”),121.0(C-1”'),110.4(C-2”',6”'),147.6(C-3”',5”'),141.2(C-4”'),166.6(C-7”')。
化合物6:丹皮酚苷F(paeonolide F)：黄白色固体；分子式：C27H32O16；分子量：612；1H-NMR(400MHz,pyridine-d5)δ:7.27(1H,d,J＝2.0Hz,H-3),6.70(1H,dd,J＝9.0,2.0Hz,H-5),8.09(1H,d,J＝9.0Hz,H-6),2.93(3H,s,CH3),3.82(3H,s,OCH3),5.74(1H,d,J＝7.8Hz,H-1'),4.33(1H,m,H-2'),6.18(1H,t,J＝7.0Hz,H-3'),4.42(1H,m,H-4'),4.39(1H,m,H-5'),4.40(1H,m,H-6'),4.80(1H,d,J＝10.2Hz,H-6'),4.85(1H,d,J＝7.0Hz,H-1”),4.44(1H,m,H-2”),4.12(1H,m,H-3”),4.29(1H,m,H-4”),3.69(1H,d,J＝11.7Hz,H-5”),4.24(1H,m,H-5”),7.84(2H,s,H-2”',6”')；13C-NMR(100MHz,pyridine-d5)δ:122.0(C-1),159.8(C-2),101.8(C-3),165.0(C-4),108.9(C-5),132.3(C-6),32.6(CH3),197.2(C＝O),55.9(OCH3),102.2(C-1'),72.7(C-2'),79.7(C-3'),77.3(C-4'),69.2(C-5'),69.7(C-6'),106.1(C-1”),72.4(C-2”),74.5(C-3”),69.5(C-4”),67.0(C-5”),121.4(C-1”'),110.5(C-2”',6”'),147.5(C-3”',5”'),140.9(C-4”'),167.2(C-7”')。
化合物7:suffruticoside B:白色固体；分子式：C27H32O16；分子量：612；1H-NMR(400MHz,pyridine-d5)δ:7.31(1H,s,H-3),6.67(1H,d,J＝8.6Hz,H-5),8.08(1H,d,J＝8.6Hz,H-6),2.94(3H,s,CH3),3.85(3H,s,OCH3),5.64(1H,d,J＝7.8Hz,H-1'),5.57(1H,d,J＝2.0Hz,H-1”),7.88(2H,s,H-2”',6”')；13C-NMR(100MHz,pyridine-d5)δ:122.4(C-1),160.3(C-2),102.8(C-3),165.3(C-4),108.7(C-5),132.7(C-6),32.8(CH3),197.5(C＝O),56.1(OCH3),102.7(C-1'),74.9(C-2'),76.4(C-3'),72.7(C-4'),75.3(C-5'),68.5(C-6'),111.6(C-1”),78.1(C-2”),80.6(C-3”),75.4(C-4”),65.8(C-5”),121.1(C-1”'),110.9(C-2”',6”'),147.9(C-3”',5”'),141.6(C-4”'),167.1(C-7”')。
化合物8:suffruticoside D:白色固体；分子式：C27H32O16；分子量：612；1H-NMR(400MHz,pyridine-d5)δ:7.31(1H,d,J＝2.0Hz,H-3),6.69(1H,dd,J＝8.4,2.0Hz,H-5),8.10(1H,d,J＝8.4Hz,H-6),2.95(3H,s,CH3),3.87(3H,s,OCH3),5.67(1H,d,J＝7.6Hz,H-1'),4.65(1H,d,J＝7.0Hz,H-1”),7.91(2H,s,H-2”',6”')；13C-NMR(100MHz,pyridine-d5)δ:122.2(C-1),160.3(C-2),102.6(C-3),165.3(C-4),109.4(C-5),132.6(C-6),32.9(CH3),197.5(C＝O),56.3(OCH3),102.0(C-1'),74.6(C-2'),75.7(C-3'),72.7(C-4'),75.7(C-5'),69.9(C-6'),106.1(C-1”),72.3(C-2”),74.9(C-3”),69.6(C-4”),67.2(C-5”),121.0(C-1”'),110.9(C-2”',6”'),147.9(C-3”',5”'),141.7(C-4”'),167.4(C-7”')。
本发明对上述酚苷类化合物进行体外抗补体活性试验测定，结果表明，所述得酚苷类化合物对补体系统的经典途径和旁路途径均具有较强的抑制作用，50％溶血所需最小供试品浓度分别为0.062-0.185mg/ml，0.102-0.393mg/ml(如表1所示)。
表1是化合物1-8对补体系统经典途径和旁路途径的抑制作用(Mean±SD,n＝3)。
表1
附图说明
图1是丹皮中酚苷类化合物1-8的提取分离流程图。
为了便于理解，以下将通过具体的附图和实施例对本发明进行详细地描述。需要特别指出的是，具体实例和附图仅是为了说明，显然本领域的普通技术人员可以根据本文说明，在本发明的范围内对本发明做出各种各样的修正和改变，这些修正和改变也纳入本发明的范围内。
具体实施方式
实施例1.制备酚苷类化合物
取丹皮20kg，粉碎，用95％乙醇室温冷浸(40L×3次)，合并提取液并浓缩得2.6kg的浸膏，取其中的1.0kg浸膏加水混悬，依次以等体积乙酸乙酯、正丁醇萃取(各2.0L×5次)，合并各萃取液并浓缩至干，得乙酸乙酯萃取物390g及正丁醇萃取物260g。取正丁醇部位浸膏加水混悬，上样于Diaion HP-20树脂，依次用水，25％、50％、75％、100％甲醇水溶液洗脱，得5个流份(PS-BU-1～5)。流份(PS-BU-2～4)分别经ODS柱色谱(甲醇：水，25:75-80:20)和Sephadex LH-20(甲醇)反复纯化，制备HPLC分离得到化合物丹皮酚苷A(paeonolideA,1)、丹皮酚苷B(paeonolide B,2)、丹皮酚苷C(paeonolide C,3)、丹皮酚苷D(paeonolideD,4)、丹皮酚苷E(paeonolide E,5)、丹皮酚苷F(paeonolide F,6)、suffruticoside B(7)、suffruticoside D(8)。其中，
化合物1:丹皮酚苷A(paeonolide A)：黄白色固体；分子式：C34H36O20；分子量：764；1H-NMR(400MHz,CD3OD)δ:6.84(1H,s,H-3),6.65(1H,d,J＝8.6Hz,H-5),7.65(1H,d,J＝8.6Hz,H-6),2.37(3H,s,CH3),3.86(3H,s,OCH3),5.60(1H,d,J＝7.0Hz,H-1'),5.50(1H,m,H-2'),5.55(1H,m,H-3'),3.82(1H,m,H-4'),3.94(1H,m,H-5'),3.72(1H,d,J＝11.0,6.3Hz,H-6'),4.10(1H,d,J＝11.0Hz,H-6'),4.98(1H,b rs,H-1”),3.94(1H,m,H-2”),3.77(1H,d,J＝9.8Hz,H-4”),4.00(1H,d,J＝9.8Hz,H-4”),3.60(2H,s,H-5”),6.94(2H,s,H-2”',6”'),7.02(2H,s,H-2””,6””)；13C-NMR(100MHz,CD3OD)δ:122.8(C-1),159.5(C-2),102.9(C-3),166.2(C-4),109.2(C-5),133.1(C-6),32.3(CH3),200.3(C＝O),56.4(OCH3),99.8(C-1'),72.9(C-2'),76.9(C-3'),69.8(C-4'),77.3(C-5'),68.4(C-6'),111.1(C-1”),78.1(C-2”),80.6(C-3”),75.1(C-4”),65.6(C-5”),120.6(C-1”'),110.3(C-2”',6”'),146.4(C-3”',5”'),140.2(C-4”'),167.1(C-7”'),121.0(C-1””),110.4(C-2””,6””),146.3(C-3””,5””),140.0(C-4””),167.8(C-7””)。
化合物2:丹皮酚苷B(paeonolide B)：黄白色固体；分子式：C34H36O20；分子量：764；1H-NMR(400MHz,CD3OD)δ:6.75(1H,d,J＝2.3Hz,H-3),6.46(1H,dd,J＝8.6,2.3Hz,H-5),7.64(1H,d,J＝8.6Hz,H-6),2.61(3H,s,CH3),3.69(3H,s,OCH3),5.08(1H,d,J＝7.4Hz,H-1'),3.53(1H,m,H-2'),3.51(1H,m,H-3'),3.32(1H,m,H-4'),3.80(1H,m,H-5'),3.60(1H,m,H-6'),4.13(1H,d,J＝10.9Hz,H-6'),5.31(1H,d,J＝1.2Hz,H-1”),5.19(1H,J＝1.2Hz,H-2”),3.95(1H,d,J＝9.8Hz,H-4”),4.24(1H,d,J＝9.8Hz,H-4”),4.41(1H,J＝11.3Hz,H-5”),4.47(1H,J＝11.3Hz,H-5”),7.06(2H,s,H-2”',6”'),7.12(2H,s,H-2””,6””)；13C-NMR(100MHz,CD3OD)δ:122.3(C-1),160.3(C-2),103.2(C-3),166.2(C-4),108.4(C-5),133.0(C-6),32.1(CH3),200.7(C＝O),56.2(OCH3),102.0(C-1'),74.9(C-2'),78.3(C-3'),71.8(C-4'),77.0(C-5'),68.4(C-6'),108.8(C-1”),80.8(C-2”),79.9(C-3”),75.8(C-4”),68.0(C-5”),120.8(C-1”'),110.5(C-2”',6”'),146.5(C-3”',5”'),140.0(C-4”'),167.5(C-7”'),121.2(C-1””),110.3(C-2””,6””),146.4(C-3””,5””),139.9(C-4””),168.1(C-7””)。
化合物3:丹皮酚苷C(paeonolide C)：黄白色固体；分子式：C27H32O16；分子量：612；1H-NMR(400MHz,pyridine-d5)δ:7.23(1H,d,J＝2.3Hz,H-3),6.61(1H,dd,J＝9.0,2.3Hz,H-5),7.95(1H,d,J＝9.0Hz,H-6),2.81(3H,s,CH3),3.85(3H,s,OCH3),5.85(1H,d,J＝8.2Hz,H-1'),6.12(1H,t,J＝9.0Hz,H-2'),4.43(1H,t,J＝9.0Hz,H-3'),4.14(1H,m,H-4'),4.34(1H,m,H-5'),4.10(1H,dd,J＝10.2,6.6Hz,H-6'),4.78(1H,d,J＝10.2Hz,H-6'),5.69(1H,d,J＝2.4Hz,H-1”),4.74(1H,J＝2.4Hz,H-2”),4.37(1H,d,J＝9.0Hz,H-4”),4.63(1H,d,J＝9.0Hz,H-4”),4.18(2H,J＝5.0Hz,H-5”),7.88(2H,s,H-2”',6”')；13C-NMR(100MHz,pyridine-d5)δ:122.1(C-1),158.9(C-2),101.9(C-3),164.7(C-4),108.3(C-5),132.3(C-6),32.5(CH3),196.9(C＝O),55.8(OCH3),99.3(C-1'),74.5(C-2'),76.5(C-3'),71.6(C-4'),77.5(C-5'),68.9(C-6'),111.2(C-1”),77.7(C-2”),80.3(C-3”),75.0(C-4”),65.4(C-5”),121.0(C-1”'),110.4(C-2”',6”'),147.6(C-3”',5”'),141.2(C-4”'),166.7(C-7”')。
化合物4:丹皮酚苷D(paeonolide D)：黄白色固体；分子式：C27H32O16；分子量：612；1H-NMR(400MHz,CD3OD)δ:6.86(1H,d,J＝2.3Hz,H-3),6.69(1H,dd,J＝8.6,2.3Hz,H-5),7.76(1H,d,J＝8.6Hz,H-6),2.63(3H,s,CH3),3.88(3H,s,OCH3),5.18(1H,d,J＝7.8Hz,H-1'),3.79(1H,m,H-2'),5.26(1H,t,J＝9.0Hz,H-3'),3.68(1H,m,H-4'),3.82(1H,m,H-5'),3.63(1H,m,H-6'),4.05(1H,d,J＝10.2Hz,H-6'),4.96(1H,d,J＝2.7Hz,H-1”),3.91(1H,J＝2.3Hz,H-2”),3.75(1H,d,J＝9.8Hz,H-4”),3.97(1H,d,J＝9.8Hz,H-4”),3.58(2H,s,H-5”),7.15(2H,s,H-2”',6”')；13C-NMR(100MHz,CD3OD)δ:122.6(C-1),160.4(C-2),103.1(C-3),166.3(C-4),109.1(C-5),133.1(C-6),32.3(CH3),200.5(C＝O),56.3(OCH3),102.4(C-1'),77.0(C-2'),79.1(C-3'),69.8(C-4'),73.3(C-5'),68.5(C-6'),111.0(C-1”),78.0(C-2”),80.5(C-3”),75.0(C-4”),65.5(C-5”),121.6(C-1”'),110.4(C-2”',6”'),146.4(C-3”',5”'),139.8(C-4”'),168.1(C-7”')。
化合物5:丹皮酚苷E(paeonolide E)：黄白色固体；分子式：C27H32O16；分子量：612；1H-NMR(400MHz,pyridine-d5)δ:7.23(1H,d,J＝2.3Hz,H-3),6.64(1H,dd,J＝9.0,2.3Hz,H-5),7.98(1H,d,J＝9.0Hz,H-6),2.83(3H,s,CH3),3.88(3H,s,OCH3),5.89(1H,d,J＝8.2Hz,H-1'),6.08(1H,t,J＝9.4Hz,H-2'),4.44(1H,m H-3'),4.24(1H,m,H-4'),4.42(1H,m,H-5'),4.32(1H,dd,J＝10.5,6.6Hz,H-6'),4.83(1H,d,J＝10.5Hz,H-6'),4.84(1H,d,J＝7.0Hz,H-1”),4.41(1H,m,H-2”),4.13(1H,m,H-3”),4.29(1H,m,H-4”),3.70(1H,d,J＝10.6Hz,H-5”),4.30(1H,m,H-5”),7.89(2H,s,H-2”',6”')；13C-NMR(100MHz,pyridine-d5)δ:122.0(C-1),159.0(C-2),101.3(C-3),164.8(C-4),109.0(C-5),132.3(C-6),32.5(CH3),197.0(C＝O),56.0(OCH3),99.2(C-1'),74.5(C-2'),76.4(C-3'),71.5(C-4'),77.8(C-5'),70.0(C-6'),106.0(C-1”),72.3(C-2”),74.4(C-3”),69.3(C-4”),66.9(C-5”),121.0(C-1”'),110.4(C-2”',6”'),147.6(C-3”',5”'),141.2(C-4”'),166.6(C-7”')。
化合物6:丹皮酚苷F(paeonolide F)：黄白色固体；分子式：C27H32O16；分子量：612；1H-NMR(400MHz,pyridine-d5)δ:7.27(1H,d,J＝2.0Hz,H-3),6.70(1H,dd,J＝9.0,2.0Hz,H-5),8.09(1H,d,J＝9.0Hz,H-6),2.93(3H,s,CH3),3.82(3H,s,OCH3),5.74(1H,d,J＝7.8Hz,H-1'),4.33(1H,m,H-2'),6.18(1H,t,J＝7.0Hz,H-3'),4.42(1H,m,H-4'),4.39(1H,m,H-5'),4.40(1H,m,H-6'),4.80(1H,d,J＝10.2Hz,H-6'),4.85(1H,d,J＝7.0Hz,H-1”),4.44(1H,m,H-2”),4.12(1H,m,H-3”),4.29(1H,m,H-4”),3.69(1H,d,J＝11.7Hz,H-5”),4.24(1H,m,H-5”),7.84(2H,s,H-2”',6”')；13C-NMR(100MHz,pyridine-d5)δ:122.0(C-1),159.8(C-2),101.8(C-3),165.0(C-4),108.9(C-5),132.3(C-6),32.6(CH3),197.2(C＝O),55.9(OCH3),102.2(C-1'),72.7(C-2'),79.7(C-3'),77.3(C-4'),69.2(C-5'),69.7(C-6'),106.1(C-1”),72.4(C-2”),74.5(C-3”),69.5(C-4”),67.0(C-5”),121.4(C-1”'),110.5(C-2”',6”'),147.5(C-3”',5”'),140.9(C-4”'),167.2(C-7”')。
化合物7:suffruticoside B:白色固体；分子式：C27H32O16；分子量：612；1H-NMR(400MHz,pyridine-d5)δ:7.31(1H,s,H-3),6.67(1H,d,J＝8.6Hz,H-5),8.08(1H,d,J＝8.6Hz,H-6),2.94(3H,s,CH3),3.85(3H,s,OCH3),5.64(1H,d,J＝7.8Hz,H-1'),5.57(1H,d,J＝2.0Hz,H-1”),7.88(2H,s,H-2”',6”')；13C-NMR(100MHz,pyridine-d5)δ:122.4(C-1),160.3(C-2),102.8(C-3),165.3(C-4),108.7(C-5),132.7(C-6),32.8(CH3),197.5(C＝O),56.1(OCH3),102.7(C-1'),74.9(C-2'),76.4(C-3'),72.7(C-4'),75.3(C-5'),68.5(C-6'),111.6(C-1”),78.1(C-2”),80.6(C-3”),75.4(C-4”),65.8(C-5”),121.1(C-1”'),110.9(C-2”',6”'),147.9(C-3”',5”'),141.6(C-4”'),167.1(C-7”')。
化合物8:suffruticoside D:白色固体；分子式：C27H32O16；分子量：612；1H-NMR(400MHz,pyridine-d5)δ:7.31(1H,d,J＝2.0Hz,H-3),6.69(1H,dd,J＝8.4,2.0Hz,H-5),8.10(1H,d,J＝8.4Hz,H-6),2.95(3H,s,CH3),3.87(3H,s,OCH3),5.67(1H,d,J＝7.6Hz,H-1'),4.65(1H,d,J＝7.0Hz,H-1”),7.91(2H,s,H-2”',6”')；13C-NMR(100MHz,pyridine-d5)δ:122.2(C-1),160.3(C-2),102.6(C-3),165.3(C-4),109.4(C-5),132.6(C-6),32.9(CH3),197.5(C＝O),56.3(OCH3),102.0(C-1'),74.6(C-2'),75.7(C-3'),72.7(C-4'),75.7(C-5'),69.9(C-6'),106.1(C-1”),72.3(C-2”),74.9(C-3”),69.6(C-4”),67.2(C-5”),121.0(C-1”'),110.9(C-2”',6”'),147.9(C-3”',5”'),141.7(C-4”'),167.4(C-7”')。
实施例2.体外抗补体经典途径试验
取补体(豚鼠血清)0.1ml，加入巴比妥缓冲液(BBS)配制成1:10的溶液，用BBS对倍稀释成1:20、1:40、1:80、1:160、1:320、1:640和1：1280的溶液。取1:1000溶血素、各浓度补体及2％羊红细胞(SRBC)各0.1ml溶于0.3ml BBS中，混匀，37℃水浴30min后放入低温高速离心机，在5000rpm、4℃条件下离心10min。分别取每管上清0.2ml于96孔板，在405nm测定其吸光度；实验同时设置全溶血组(0.1ml 2％SRBC溶于0.5ml三蒸水)；以三蒸水溶血管的吸光度作为全溶血标准，计算溶血率。以补体稀释度为X轴，溶血百分率为Y轴作图，选择达到相似高溶血率的最低补体浓度作为确保体系能正常溶血所需的临界补体浓度，取临界浓度的补体与供试品混匀，加入适量BBS、溶血素和2％SRBC，37℃水浴30min后放入低温高速离心机，5000rpm、4℃条件下离心10min后分别取每管上清0.2ml于96孔板，405nm下测定吸光度；实验同时设置供试品对照组、补体组和全溶血组；将供试品吸光度值扣除相应供试品对照组吸光度值后计算溶血率，以供试品浓度作为X轴，溶血抑制率作为Y轴作图，计算50％抑制溶血所需供试品的浓度(CH50)。
实施例3.体外抗补体旁路途径试验
取补体(人血清)0.2ml，加入AP稀释液(巴比妥缓冲液，pH＝7.4,含5mM Mg2+,8mMEGTA)配制成1:5的溶液，并对倍稀释成1:10、1:20、1:40、1:80、1:160、1:320和1:640的溶液。取各浓度补体0.15ml、AP稀释液0.15ml及0.5％兔红细胞(RE)0.20ml，混匀，37℃水浴30min后置于低温高速离心机，在5000rpm、4℃条件下离心10min。分别取每管上清0.2ml于96孔板，在405nm测定吸光度。实验同时设置全溶血组(0.20ml 0.5％RE溶于0.3ml三蒸水)。以三蒸水溶血管的吸光度作为全溶血标准，计算溶血率。以补体稀释度为X轴，溶血百分率为Y轴作图。选择达到相似高溶血率的最低补体浓度作为确保体系能正常溶血所需的临界补体浓度。取确定的临界浓度的补体与供试品混匀，于37℃预水浴10min后，加入0.2ml0.5％RE。将每管37℃水浴30min后置于低温高速离心机，5000rpm、4℃条件下离心10min后，分别取每管上清0.2ml于96孔板，405nm下测定其吸光度。实验同时设置供试品对照组、补体组和全溶血组。将供试品吸光度值扣除相应供试品对照组吸光度值后计算溶血率。以供试品浓度作为X轴，溶血抑制率作为Y轴作图，计算50％抑制溶血所需供试品的浓度(AP50)
试验结果表明，所述的酚苷类化合物对补体系统的经典途径和旁路途径均具有较强的抑制作用，50％溶血所需最小供试品浓度分别为0.062-0.185mg/ml，0.102-0.393mg/ml(如表1所示)。
表1是化合物1-8对补体系统经典途径和旁路途径的抑制作用(Mean±SD,n＝3)。
表1
本发明中实验采用的试剂均为本领域公知技术，可市购。
Claims (4)
1.具有以下结构通式的活性酚苷类化合物：
当R1＝R2＝G,R3＝H,R4＝AP,化合物为丹皮酚苷A(paeonolide A)；
当R1＝R2＝R3＝H,R4＝GAP,化合物为丹皮酚苷B(paeonolide B)；
当R1＝G,R2＝R3＝H,R4＝AP,化合物为丹皮酚苷C(paeonolide C)；
当R1＝R3＝H,R2＝G,R4＝AP,化合物为丹皮酚苷D(paeonolide D)；
当R1＝R3＝H,R2＝G,R4＝A,化合物为丹皮酚苷E(paeonolide E)；
当R1＝G,R2＝R3＝H,R4＝A,化合物为丹皮酚苷F(paeonolide F)。
2.按权利要求1所述的酚苷类化合物，其特征在于，通过下述方法制备：
取丹皮，粉碎，用95％乙醇室温冷浸，提取液减压回收溶剂得到浸膏，取其中浸膏加水混悬，依次用乙酸乙酯、正丁醇萃取，各萃取液浓缩至干，得乙酸乙酯萃取物及正丁醇萃取物；取正丁醇部位浸膏加水混悬，上样于Diaion HP-20树脂，依次用水，25％、50％、75％、100％甲醇水溶液洗脱，所得流份经ODS柱色谱和Sephadex LH-20反复纯化，制备HPLC分离得到化合物丹皮酚苷A、丹皮酚苷B、丹皮酚苷C、丹皮酚苷D、丹皮酚苷E、丹皮酚苷F。
3.权利要求1所述的酚苷类化合物在制备抗补体药物中的用途。
4.按权利要求3所述的用途，所述的酚苷类化合物对补体系统经典途径和旁路途径均有抑制作用。
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| CN1903259A (zh) * | 2005-07-29 | 2007-01-31 | 天津天士力现代中药研究开发有限公司 | 一种丹皮药材的提取分离方法 |
| WO2007106049A1 (en) * | 2006-03-16 | 2007-09-20 | Moleac Pte Ltd | Combination therapy for treatment of patients with neurological disorders and cerebral infarction |
| CN101181373A (zh) * | 2007-12-07 | 2008-05-21 | 戴敏 | 丹皮有效部位药物组合物、其制备方法及应用 |
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2013
- 2013-06-06 CN CN201310222851.6A patent/CN104231012B/zh not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1903259A (zh) * | 2005-07-29 | 2007-01-31 | 天津天士力现代中药研究开发有限公司 | 一种丹皮药材的提取分离方法 |
| WO2007106049A1 (en) * | 2006-03-16 | 2007-09-20 | Moleac Pte Ltd | Combination therapy for treatment of patients with neurological disorders and cerebral infarction |
| CN101181373A (zh) * | 2007-12-07 | 2008-05-21 | 戴敏 | 丹皮有效部位药物组合物、其制备方法及应用 |
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| Characterization of compounds in the Chinese herbal drug Mu-Dan-Pi by liquid chromatography coupled to electrospray ionization mass spectrometry;Shun-jun Xu，等;《RAPID COMMUNICATIONS IN MASS SPECTROMETRY》;20061017;第20卷(第22期);3275–3288 * |
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