2016, Vol. 47
引用本文 | doi: 10.7501/j.issn.0253-2670.2016.06.004 |
皱盖假芝Amauroderma rude (Berk.) Pat. 为担子菌纲灵芝科（Ganodermataceae）假芝属Amauroderma大型真菌，生于阔叶树桩旁地上，分布于我国福建、海南、贵州等省区[1]。皱盖假芝是海南省主要的药用灵芝科真菌资源[2]，在民间用于提高免疫和肿瘤的辅助治疗。有关皱盖假芝的研究报道极少，只对皱盖假芝深层发酵液多糖提取工艺进行了研究[3]，迄今关于皱盖假芝的小分子天然产物及其生物活性研究未见报道，其小分子药效物质基础尚不清楚。近年灵芝科真菌的药效物质基础与药理作用的研究方兴未艾，研究的真菌种类越来越多，其新颖活性成分和新的药理功效仍不断被发现[4, 5, 6, 7, 8]。为了深入认识皱盖假芝中化学成分及其生物活性，本实验采用各种色谱分离方法从皱盖假芝子实体的乙醇提取物中分离鉴定了12个化合物，分别鉴定为diptoindonesin D（1）、6-脱氧巴西红厚壳素（6-deoxyjacareubin，2）、巴西红厚壳素（jacareubin，3）、3-羧基吲哚（1H-indole-3-carboxylic acid，4）、3,4-二羟基苯甲酸甲酯（methyl-3,4-dihydroxybenzoate，5）、3,4-二羟基苯乙醇（3,4-dihydroxyphenylethanol，6）、3β-羟基-7,22E-二烯-麦角甾（3β-hydroxy-7,22- dien-ergosta，7）、3β,7α-二羟基-8,22E-二烯-5α,6α-环氧-麦角甾（3β,7α-dihydroxy-8,22E-5α,6α- epoxyergosta，8）、3β-羟基-7α-甲氧基-8(14),22E-二烯-5α,6α-环氧-麦角甾[3β-hydroxy-7α-methoxy-8 (14),22E-dien-5α,6α-epoxy-ergosta，9]、5α,8α-过氧麦角固醇（ergosterol 5α,8α-peroxide，10）、3β,5β,7β-三羟基-6,22E-二烯-麦角甾（3β-5β-8β-trihydroxy- 6,22E-ergosta，11）、3β,5α,6β-三羟基-7,22E-二烯-麦角甾（3β,5α,6β-trihydroxy-7,22E-dien-ergosta，12）。所有化合物均为首次从皱盖假芝子实体中分离得到。采用MTT法测定抗肿瘤活性发现化合物2、3和9具有一定肿瘤细胞毒活性。
1 仪器与材料核磁共振（NMR）用Bruker DRX-500核磁共振光谱仪测定；质谱（MS）用Bruker HCT/Esquire型液相离子阱色谱质谱联用仪测定；柱色谱硅胶（200～300目）和薄层色谱硅胶GF254均为青岛海洋化工厂生产；Sephadex LH-20为GE Biosciences公司产品。
皱盖假芝于2011年9月采自海南省陵水县，由海南大学吴兴亮教授鉴定为灵芝科假芝属真菌皱盖假芝Amauroderma rude (Berk.) Pat.，凭证标本（2011JZ02）存放于中国热带农业科学院热带生物技术研究所。
2 提取与分离皱盖假芝子实体（5.0 kg）晒干后粉碎，用95%乙醇浸提3次，每次7 d。滤液经真空减压浓缩至无醇味后得粗浸膏，粗浸膏分散于水中成悬浊液，依次用醋酸乙酯、正丁醇萃取，萃取液分别减压浓缩至浸膏，得醋酸乙酯部分64 g和正丁醇部分18 g。醋酸乙酯部分经减压硅胶柱色谱分离，以石油醚-醋酸乙酯（8∶1→1∶5）梯度洗脱，得到5个组分Fr. 1～5。Fr. 1（18 g）采用硅胶柱色谱，以石油醚-醋酸乙酯（6∶1→3∶1）梯度洗脱和重结晶得到化合物7（400 mg）。Fr. 2（4.4 g）采用硅胶柱色谱，以石油醚-醋酸乙酯（6∶1→3∶1）梯度洗脱和Sephadex LH-20色谱（95%乙醇）得到化合物2（15 mg）。Fr. 3（2.5 g）采用硅胶柱色谱，以石油醚-醋酸乙酯（3∶1→2∶1）梯度洗脱和Sephadex LH-20色谱（氯仿-甲醇1∶1）得到化合物3（18 mg）、4（4 mg）、5（11.5 mg）。Fr. 4（1.4 g）采用硅胶柱色谱，以石油醚-醋酸乙酯（2∶1→1∶1）梯度洗脱和Sephadex LH-20色谱（氯仿-甲醇1∶1）得到化合物1（3.7 mg）、6（7 mg）、8（12 mg）、9（27 mg）、10（300 mg）。正丁醇部分以氯仿-甲醇（7∶1→3∶1）梯度洗脱和Sephadex LH-20色谱（氯仿-甲醇1∶1）得到化合物11（15 mg）和12（16 mg）。
3 结构鉴定化合物1：白色无定型粉末；ESI-MS m/z: 379 [M＋H]+，分子式C21H15O7。1H-NMR (500 MHz,CDCl3) δ: 6.84 (1H,d,J = 2.6 Hz,H-6),6.77 (1H,d,J = 2.5 Hz,H-14),6.62 (1H,d,J = 2.5 Hz,H-12),6.60 (2H,d,J = 8.0 Hz,H-18,20),6.84 (2H,d,J = 6.5 Hz,H-17,21),6.32 (1H,d,J = 2.6 Hz,H-4),5.82 (1H,s,H-9)；13C-NMR (125 MHz,CDCl3) δ: 198.7 (C-1),112.0 (C-2),167.3 (C-3),107.2 (C-4),165.5 (C-5),113.3 (C-6),139.8 (C-7),198.1 (C-8),55.6 (C-9),110.0 (C-10),143.1 (C-15),111.2 (C-14),157.5 (C-13),107.4 (C-12),159.3 (C-11),131.0 (C-16),129.2 (C-17,21),116.3 (C-18,20),157.1 (C-19)。以上数据与文献报道基本一致[9]，故鉴定化合物1为diptoindonesin D。
化合物2：淡黄色粉末，ESI-MS m/z: [M＋Na]+ 333，分子式C18H14O5。1H-NMR (500 MHz,DMSO-d6) δ: 13.12 (1H,s,1-OH),7.61 (1H,dd,J = 7.8,1.8 Hz,H-8),7.21 (1H,dd,J = 7.9,1.8 Hz,H-6),7.20 (1H,dd,J = 7.9,7.8 Hz,H-7),6.79 (1H,d,J = 10.0 Hz,H-13),6.43 (1H,s,H-4),5.60 (1H,d,J = 10.0 Hz,H-14),1.45 (6H,s,H-15,16)；13C-NMR (125 MHz,DMSO-d6) δ: 161.6 (C-1),105.2 (C-2),158.0 (C-3),95.8 (C-4),146.5 (C-5),121.1 (C-6),124.5 (C-7),116.0 (C-8),181.9 (C-9),78.9 (C-12),115.7 (C-13),128.4 (C-14),28.6 (C-15),28.6 (C-16),157.7 (C-4a),121.9 (C-8a),104.1 (C-9a),146.0 (C-10a)。以上数据与文献报道基本一致[10]，故鉴定化合物2为6-脱氧巴西红厚壳素。
化合物3：淡黄色粉末，ESI-MS m/z: [M＋Na]+ 349，分子式C18H14O6。1H-NMR (500 MHz,DMSO-d6) δ: 13.43 (1H,s,1-OH),7.40 (1H,d,J = 8.7 Hz,H-8),6.84 (1H,d,J = 8.7 Hz,H-7),6.40 (1H,d,J = 10.1 Hz,H-13),6.30 (1H,s,H-4),5.65 (1H,d,J = 10.1 Hz,H-14),1.33 (6H,s,H-15,16)；13C-NMR (125 MHz,DMSO-d6) δ: 159.6 (C-1),103.9 (C-2),157.0 (C-3),94.8 (C-4),132.6 (C-5),152.2 (C-6),113.3 (C-7),116.0 (C-8),180.0 (C-9),78.3 (C-12),114.6 (C-13),128.4 (C-14),28.0 (C-15,16),156.6 (C-4a),113.0 (C-8a),102.3 (C-9a),146.1 (C-10a)。以上数据与文献报道一致[11]，故鉴定化合物3为巴西红厚壳素。
化合物4：无色油状物，ESI-MS m/z: 162 [M＋H]+，分子式C9H7NO2。1H NMR (500 MHz,CD3OD) δ: 7.98 (1H,s,H-2),8.09 (1H,d,J = 7.2 Hz,H-4),7.20 (1H,m,H-5),7.18 (1H,m,H-6),7.45 (1H,d,J = 7.2 Hz,H-7)；13C-NMR (125 MHz,CD3OD) δ: 169.2 (COOH),133.4 (C-2),127.6 (C-3),122.4 (C-4),123.6 (C-5),122.0 (C-6),112.9 (C-7),108.7 (C-3a),138.2 (C-7a)。以上数据与文献报道基本一致[12]，故鉴定化合物4为3-羧基吲哚。
化合物5：无定形粉末，ESI-MS m/z: 191 [M＋Na]+，分子式C8H8O4。1H-NMR (500 MHz,CD3OD) δ: 6.81 (1H,d,J = 2.0 Hz,H-2),7.40 (1H,dd,J = 8.3,2.0 Hz,H-6),7.42 (1H,d,J = 8.3 Hz,H-5),3.83 (3H,s,OCH3)；13C-NMR (125 MHz,CD3OD) δ: 122.6 (C-1),115.8 (C-2),151.7 (C-3),146.2 (C-4),123.6 (C-5),117.4 (C-6),168.9 (C-7),52.3 (OCH3)。上述数据与文献报道基本一致[13]，故鉴定化合物5为3,4-二羟基苯甲酸甲酯。
化合物6：无定形粉末，ESI-MS m/z: 177 [M＋Na]+，分子式C8H10O3。1H-NMR (500 MHz,CD3OD) δ: 6.70 (2H,m,H-5,6),6.55 (1H,d,J = 2.0 Hz,H-2),3.80 (2H,t,J = 7.3 Hz,H-8),2.68 (2H,t,J = 7.3 Hz,H-7)；13C-NMR (125 MHz,CD3OD) δ: 131.8 (C-1),116.3 (C-2),146.1 (C-3),144.6 (C-4),117.1 (C-5),121.2 (C-6),39.6 (C-7),64.6 (C-8)。以上数据与文献报道基本一致[13]，故鉴定化合物6为3,4-二羟基苯乙醇。
化合物7：白色粉末，ESI-MS m/z: 421 [M＋Na]+，分子式C28H46O。1H-NMR (500 MHz,CDCl3) δ: 5.20 (1H,m,H-23),5.19 (1H,m,H-22),5.16 (1H,m,H-7),3.58 (1H,m,H-3),1.00 (3H,d,J = 6.7 Hz,H-21),0.90 (3H,d,J = 6.9 Hz,H-26),0.83 (3H,d,J = 7.0 Hz,H-27),0.82 (3H,d,J = 7.2 Hz,H-28),0.79 (3H,s,H-19),0.54 (3H,s,H-18)；13C-NMR (125 MHz,CDCl3) δ: 37.3 (C-1),29.8 (C-2),71.2 (C-3),38.1 (C-4),40.1 (C-5),31.6 (C-6),117.6 (C-7),139.7 (C-8),49.6 (C-9),34.4 (C-10),21.7 (C-11),39.6 (C-12),43.4 (C-13),55.3 (C-14),23.1 (C-15),28.2 (C-16),56.1 (C-17),12.2 (C-18),13.2 (C-19),40.6 (C-20),21.3 (C-21),135.8 (C-22),132.0 (C-23),43.0 (C-24),33.2 (C-25),20.1 (C-26),19.8 (C-27),17.7 (C-28)。以上数据与文献报道基本一致[14]，故鉴定化合物7为3β-羟基-7,22E-二烯-麦角甾。
化合物8：白色粉末，ESI-MS m/z: 451 [M＋Na]+，分子式C28H44O3。1H-NMR (500 MHz,CDCl3) δ: 5.18 (2H,m,H-22,23),4.40 (1H,d,J = 2.0 Hz,H-7),4.22 (1H,m,H-3),3.30 (1H,d,J = 2.0,Hz,H-6),1.13 (3H,s,H-19),1.01 (3H,d,J = 6.5 Hz,H-21),0.90 (3H,d,J = 6.8 Hz,H-28),0.84 (3H,d,J = 7.6 Hz,H-27),0.81 (3H,d,J = 7.5 Hz,H-26)；0.56 (3H,s,H-18)；13C-NMR (125 MHz,CDCl3) δ: 30.9 (C-1),30.3 (C-2),68.7 (C-3),39.3 (C-4),65.8 (C-5),62.7 (C-6),67.2 (C-7),134.6 (C-8),127.0 (C-9),38.1 (C-10),23.5 (C-11),35.8 (C-12),42.2 (C-13),49.7 (C-14),24.0 (C-15),29.1 (C-16),53.8 (C-17),11.4 (C-18),23.0 (C-19),40.5 (C-20),21.1 (C-21),135.7 (C-22),132.1 (C-23),43.0 (C-24),33.2 (C-25),19.8 (C-26),20.1 (C-27),17.7 (C-28)。以上数据与文献报道基本一致[15]，故鉴定化合物8为3β,7α-二羟基-8,22E-二烯-5α,6α-环氧-麦角甾。
化合物9：无色油状物，ESI-MS m/z: 465 [M＋Na]+，分子式C29H46O3。1H-NMR (500 MHz,CDCl3) δ: 5.24 (1H,m,H-23),5.16 (1H,m,H-22),4.20 (1H,d,J = 3.3 Hz,H-7),3.76 (1H,m,H-3),3.43 (3H,s,7-OCH3),3.11 (1H,d,J = 3.0 Hz,H-6),1.05 (3H,d,J = 6.6 Hz,H-21),0.96 (3H,d,J = 6.8 Hz,H-28),0.89 (3H,s,H-19),0.86 (6H,d,J = 6.5 Hz,H-26,27),0.54 (3H,s,H-18)；13C-NMR (125 MHz,CDCl3) δ: 33.4 (C-1),31.9 (C-1),69.2 (C-3),40.4 (C-4),66.9 (C-5),59.3 (C-6),74.3 (C-7),124.1 (C-8),41.6 (C-9),37.0 (C-10),20.3 (C-11),37.9 (C-12),55.1 (C-13),154.0 (C-14),25.8 (C-15),28.4 (C-16),58.2 (C-17),18.7 (C-18),17.0 (C-19),40.6 (C-20),21.8 (C-21),136.8 (C-22),133.4 (C-23),44.4 (C-24),34.4 (C-25),20.5 (C-26),20.2 (C-27),18.2 (C-28),54.8 (OCH3)。上述数据与文献报道一致[16]，故鉴定化合物9为3β-羟基-7α-甲氧基-8(14),22E-二烯-5α,6α-环氧-麦角甾。
化合物10：白色粉末，ESI-MS m/z: 451 [M＋Na]+，分子式C28H44O3。1H-NMR (500 MHz,CDCl3) δ: 6.49 (1H,d,J = 8.5 Hz,H-7),6.23 (1H,d,J = 8.5 Hz,H-6),5.22 (1H,dd,J = 7.6,15.2 Hz,H-23),5.14 (1H,dd,J = 8.2,15.2 Hz,H-22),3.97 (1H,m,H-3),0.99 (3H,d,J = 6.6 Hz,H-21),0.92 (3H,d,J = 6.5 Hz,H-28),0.88 (3H,s,H-19),0.62 (3H,s,H-18),0.81 (6H,d,J = 6.0 Hz,H-26,27)；13C-NMR (125 MHz,CDCl3) δ: 37.1 (C-1),30.2 (C-2),66.6 (C-3),32.8 (C-4),82.3 (C-5),135.3 (C-6),130.9 (C-7),79.5 (C-8),51.3 (C-9),37.1 (C-10),21.8 (C-11),39.5 (C-12),44.7 (C-13),51.8 (C-14),23.5 (C-15),28.8 (C-16),56.3 (C-17),13.0 (C-18),18.3 (C-19),39.9 (C-20),21.0 (C-21),135.6 (C-22),132.4 (C-23),42.9 (C-24),33.2 (C-25),19.8 (C-26),20.1 (C-27),17.7 (C-28)。以上数据与文献报道基本一致[17]，故鉴定化合物10为5α,8α-过氧麦角固醇。
化合物11：白色粉末，ESI-MS m/z: 453 [M＋Na]+，分子式C28H46O3。1H-NMR (500 MHz,CDCl3) δ: 5.14 (1H,s,H-7),5.08 (1H,dd,J = 7.5,15.3 Hz,H-23),5.00 (1H,dd,J = 6.0,15.3 Hz,H-22),3.66 (1H,m,H-3),0.98 (3H,d,J = 6.5 Hz,H-21),0.91 (3H,d,J = 6.2 Hz,H-28),0.87 (3H,s,H-19),0.76 (3H,d,J = 6.5 Hz,H-26),0.66 (3H,d,J = 6.5 Hz,H-27),0.55 (1H,s,H-18)；13C-NMR (125 MHz,CDCl3) δ: 38.8 (C-1),30.3 (C-2),67.1 (C-3),36.8 (C-4),75.8 (C-5),143.1 (C-6),117.4 (C-7),73.0 (C-8),43.0 (C-9),32.6 (C-10),21.8 (C-11),38.8 (C-12),43.5 (C-13),54.6 (C-14),22.8 (C-15),27.8 (C-16),55.8 (C-17),12.0 (C-18),18.0 (C-19),40.3 (C-20),20.9 (C-21),135.4 (C-22),131.4 (C-23),42.7 (C-24),32.9 (C-25),19.4 (C-26),19.7 (C-27),17.4 (C-28)。以上数据与文献报道基本一致[17]，故鉴定化合物11为3β,5β,7β-三羟基-6,22E-二烯-麦角甾。
化合物12：白色粉末，ESI-MS m/z: 453 [M＋Na]+，分子式C28H46O3。1H-NMR (500 MHz,CDCl3) δ: 5.22 (1H,d,J = 5.2 Hz,H-7),5.11 (1H,m,H-23),5.10 (1H,m,H-22),3.91 (1H,m,H-3),3.49 (1H,d,J = 5.0 Hz,H-6),0.99 (3H,s,H-19),0.93 (3H,d,J = 6.5 Hz,H-21),0.83 (3H,d,J = 6.8 Hz,H-28),0.72 (6H,d,J = 6.4 Hz,H-26,27),0.55 (3H,s,H-18)；13C- NMR (125 MHz,CD3OD) δ: 30.2 (C-1),32.7 (C-2),67.1 (C-3),39.2 (C-4),76.0 (C-5),73.0 (C-6),117.3 (C-7),143.4 (C-8),43.1 (C-9),37.0 (C-10),22.1 (C-11),38.7 (C-12),43.6 (C-13),54.7 (C-14),22.9 (C-15),27.9 (C-16),56.1 (C-17),12.2 (C-18),18.4 (C-19),40.4 (C-20),21.0 (C-21),135.5 (C-22),132.1 (C-23),42.7 (C-24),33.1 (C-25),19.8 (C-26),19.6 (C-27),17.4 (C-28)。上述数据与文献报道基本一致[18]，故鉴定化合物12为3β,5α-6β-三羟基-7,22E-二烯-麦角甾。
4 抗肿瘤活性采用MTT法[19]检测化合物2、3和7～10对5种人体肿瘤细胞（白血病细胞HL-60、肝癌细胞SMMC-7721、肺癌细胞A549、乳腺癌细胞MCF-7、结肠癌细胞SW-480）的增殖抑制活性。结果显示化合物2和3对5种人体肿瘤细胞具有一定的细胞毒活性，IC50值分别为19.59、32.25、25.87、22.76、19.90 μmol/L和7.49、9.04、4.44、21.61、14.02 μmol/L。化合物9对3种人体肿瘤细胞HL-60、MCF-7和SW-480表现一定的细胞毒活性，其IC50值分别15.98、16.28、13.26 μmol/L。化合物7、8和10对这5种肿瘤细胞无明显细胞毒活性。
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