蓝桉果实的酚性成分研究

2021-09-23王玲莉张冬丽唐生安

中草药 2021年18期

于欢，李敏，张旭，王玲莉，王佳，张冬丽，许嵘，唐生安*

1.天津医科大学药学院，天津市临床药物关键技术重点实验室，天津 300070

2.泉州医学高等专科学校药学院，福建泉州 362011

蓝桉Eucalyptus globulusLabill.为桃金娘科（Myrtaceae）桉属EucalyptusL.Herit植物，原产于澳大利亚，在我国广西、云南、四川等地均有栽培，其叶、根皮及果实均可入药[1-2]，是一种重要的药用资源。蓝桉的果实因成熟后其形状酷似倒挂的“小钟”又俗称“一口钟”，民间俗称扣子七、红喇叭花、胜利果等[3]，是我国传统民间中草药，也是蓝桉临床上常用的有效部位。一口钟富含多种挥发油、倍半萜、三萜、间苯三酚衍生物、黄酮和鞣质等化学成分[4-5]，具有抗肿瘤、抗菌、抗病毒、抗氧化和降血糖[6]等多种生物活性。

一口钟在我国分布广泛，资源丰富。本课题组前期对一口钟石油醚萃取物进行化学成分研究，发现其主要含有倍半萜和三萜类化合物[7]，在此基础上，为了进一步探索其活性成分，本实验继续对一口钟石油醚萃取物进行研究，采用硅胶柱色谱、凝胶柱色谱、以及高效液相色谱技术进行分离，采用波谱学方法与理化性质相结合鉴定了15个化合物，分别为大黄酚（chrysophanol，1）、eucarobustol E（2）、eucarobustol G（3）、eucalyptal A（4）、eucalyptal C（5）、eucalyptin C（6）、eucalyptin D（7）、eucalyptin E（8）、eucalrobusone A（9）、eucalrobusone C（10）、eucalrobusone F（11）、eucalrobusone U（12）、eucalyptone（13）、euglobal-Ia1（14）、euglobal-Ia2（15）。分离得到的化合物均为酚性成分。其中，化合物1为首次从桉属植物中分离得到的，化合物2、3为首次从该植物中分离得到，化合物9～15为首次从蓝桉果实中分离得到。其中化合物6、7、10、12、13对MCF-7细胞具有明显细胞毒活性，20 μmol/L的浓度下抑制率分别为71.83%、71.11%、70.00%、80.53%、79.87%，化合物12对MCF-7显示出较强的细胞毒活性，其半数抑制浓度（IC50）为13.84 μmol/L。

1 仪器与材料

Bruker AM-400/500/600 核磁共振仪（Bruker 公司，瑞士）；Alliance 2695 Quattro Micro TM ESI 液质联用色谱仪（Waters公司，美国）；LC3000型高效液相色谱仪（北京创新通恒科技有限公司）；YMC-Pack ODS-A制备型HPLC色谱柱（250 mm×20 mm）；Sephadex LH-20（美国GE公司），柱色谱硅胶（100～200、200～300、300～400目）和薄层板色谱硅胶GF254均为青岛海洋化工有限公司产品；实验所用试剂均为分析纯有机试剂，为天津市津东天正精细化学试剂厂产品；氘代试剂均为Cambrige Isotope Laboratories Inc.USA生产；IMark型全自动酶标仪（BIO-RAD，日本）。

一口钟购于河北安国药材批发市场，产地为云南省，经天津医科大学药学院唐生安副教授鉴定为桉树植物蓝桉Eucalyptus globulusLabill.的干燥成熟果实，标本（编号D20151011）现存放于天津医科大学药学院天然药化实验室。

2 提取与分离

将一口钟药材（干质量20 kg）粉碎，95%乙醇浸渍，每次浸渍3 d，一共浸渍5次，将浸渍液合并后减压浓缩，得到乙醇提取物的浸膏（2992 g）。将乙醇提取物用水混悬，依次用石油醚、醋酸乙酯萃取3次，分别得到石油醚萃取物（1592 g）、醋酸乙酯萃取物（406 g）。

取石油醚萃取物800 g，经硅胶柱色谱初步分离，石油醚：醋酸乙酯（60∶1→1∶1）梯度洗脱，得到16个组分Fr.1～16。Fr.6分别用石油醚-醋酸乙酯（30∶1、15∶1、8∶1、4∶1、2∶1）进行梯度洗脱分离得到14个亚组分Fr.6.1～6.14。Fr.6.3（11 g）通过ODS柱色谱分离，甲醇-水（80∶20、90∶10、95∶5、98∶2）梯度洗脱，共得到16个亚组分Fr.6.3.1～6.3.16。Fr.6.3.1（187.6 mg）经半制备HPLC（ODS-A，甲醇-水88∶12，0.3%醋酸，5 mL/min）分离，纯化得到化合物1（13.3 mg，tR＝39.1 min）；Fr.6.3.9（672.9 mg）经半制备HPLC（ODS-A，甲醇-水88∶12，0.3%醋酸，5 ml/min）分离，纯化得到化合物12（76.9 mg，tR＝182.0 min）和3（26.3 mg，tR＝194.2 min）；Fr.6.3.11（105.9 mg）过半制备液相色谱柱（ODS-A，甲醇-水93∶7，0.3%醋酸，5 mL/min），纯化得到化合物9（20.2 mg，tR＝263.9 min）、10（73.0 mg，tR＝204.0 min）。将Fr.6.8.2（517.9 mg）过半制备液相色谱柱（ODS-A，甲醇-水90∶10，0.3%醋酸），纯化得到化合物11（13.1 mg，tR＝60.5 min）。Fr.6.8（7 g）进行硅胶柱分离（二氯甲烷-甲醇-醋酸1000∶5∶3），得到17个亚组分Fr.6.8.1～6.8.17，将组分Fr.6.8.6与Fr.6.8.7合并，过半制备HPLC（ODS-A，甲醇-水89∶11，0.3%醋酸，5 mL/min）分离，得到化合物6（7.9 mg，tR＝49.7 min）和8（8.2 mg，tR＝46.7 min）。Fr.6.9（1 g）过正相硅胶柱（二氯甲烷-甲醇4∶1，0.3% 醋酸）得到化合物2（94.5 mg）。

Fr.14（59 g）组分，进行硅胶柱分离 [二氯甲烷-甲醇-乙酸（80∶1∶1）]，得到14个亚组分Fr.14.1～14.14。Fr.14.4（26 g）过正相硅胶柱色谱[二氯甲烷-甲醇（10∶1）]分离，得到19个亚组分Fr.14.4.1～14.4.19，组分Fr.14.4.2（140.6 mg）通过半制备HPLC [ODS-A，甲醇-水（88∶12），0.3% 醋酸，5 mL/min]分离，得到化合物5（4.8 mg，tR＝118.3 min）。Fr.14.5与Fr.14.6合并，经硅胶柱色谱分离，石油醚-醋酸乙酯（100∶1→1∶1）梯度洗脱得到19个亚组分Fr.14.6.1～14.6.19，Fr.14.6.8和Fr.14.6.19分别用半制备HPLC纯化 [ODS-A，甲醇-水（86∶14），0.5%醋酸，5 mL/min]，依次得到化合物4（35.8 mg）、13（264.3 mg）。

Fr.15经Toyopearl HW-40凝胶柱色谱 [二氯甲烷-甲醇（2∶1）]分离，得到6个亚组分Fr.15.1～15.6，其中Fr.15.1经硅胶柱色谱，以石油醚-二氯甲烷-醋酸乙酯-醋酸（40∶30∶1∶1）作为流动相进行分离，得到化合物14（27.5 mg）、15（17.4 mg）。

3 结构鉴定

化合物1：黄色粉末。ESI-MSm/z: 255 [M＋H]+，分子式为C15H10O4。1H-NMR (400 MHz,pyridine-d5)δ: 12.11 (1H,s,H-8),12.01 (1H,s,1-OH),7.82 (1H,d,J= 7.2 Hz,H-5),7.67 (1H,m,H-6),7.29 (1H,d,J= 8.4 Hz,H-7),7.10 (1H,s,2-OH),2.47 (3H,s,H-CH3)；13C-NMR (100 MHz,pyridine-d5)δ: 162.7 (C-1),119.9 (C-2),149.4 (C-3),121.4 (C-4),124.6 (C-5),133.3 (C-6),124.4 (C-7),162.5 (C-8),192.6 (C-9),182.0 (C-10),137.0 (C-4a),115.9 (C-8a),113.8 (C-9a),133.7 (C-10a),22.3 (C-CH3)。上述波谱数据与文献值基本一致[8]，故鉴定化合物1为大黄酚。

化合物2：浅黄色粉末。ESI-MSm/z: 487 [M＋H]+，分子式为C29H42O6。1H-NMR (400 MHz,pyridine-d5)δ: 10.54 (1H,s,8′-CHO),10.51 (1H,s,7′-CHO),3.74 (1H,dd,J= 12.8,4.4 Hz,H-9′),3.20 (3H,s,H-OCH3),2.59 (1H,dd,J= 12.8,4.0 Hz,H-10′a),2.29 (1H,dd,J= 10.0,4.4 Hz,H-1),1.25 (3H,s,H-14),1.19 (3H,overlapped,H-12),1.19 (3H,overlapped,H-13),0.99 (3H,d,J= 6.8 Hz,H-13′),0.94 (3H,overlapped,H-15),0.94 (3H,overlapped,H-12′)；13C-NMR (100 MHz,pyridine-d5)δ: 50.3 (C-1),24.4 (C-2),35.7 (C-3),48.7 (C-4),43.9 (C-5),27.9 (C-6),26.1 (C-7),20.4 (C-8),37.6 (C-9),79.6 (C-10),19.9 (C-11),29.1 (C-12),17.8 (C-13),18.6 (C-14),21.5 (C-15),171.4 (C-1′),106.6 (C-2′),171.3 (C-3′),106.6 (C-4′),170.6 (C-5′),108.6 (C-6′),192.3 (C-7′),192.0 (C-8′),35.6 (C-9′),34.6 (C-10′),27.9 (C-11′),24.8 (C-12′),22.4 (C-13′),47.9 (C-OCH3)。上述波谱数据与文献报道基本一致[9]，故鉴定化合物2为eucarobustol E。

化合物3：无定形粉末。ESI-MSm/z: 453 [MH]-，确定其分子式为C28H38O5。1H-NMR (400 MHz,pyridine-d5)δ: 10.55 (1H,s,8′-CHO),10.54 (1H,s,7′-CHO),5.22 (1H,s,H-2),3.58 (1H,dd,J= 11.6,3.6 Hz,H-9′),2.45 (1H,d,J= 10.0 Hz,H-10′a),1.48 (1H,d,J= 4.0 Hz,H-2),1.44 (1H,d,J= 3.2 Hz,H-10′b),1.42 (3H,s,H-15),1.20 (3H,overlapped,H-12),1.20 (3H,overlapped,H-13),1.05 (3H,overlapped,H-14),1.05 (3H,overlapped,H-12′),1.00 (3H,d,J= 6.6 Hz,H-13′)；13C-NMR (100 MHz,pyridine-d5)δ: 155.1 (C-1),121.7 (C-2),43.2 (C-3),52.7 (C-4),53.8 (C-5),30.3 (C-6),30.7 (C-7),26.3 (C-8),39.4 (C-9),39.6 (C-10),22.3 (C-11),30.2 (C-12),18.2 (C-13),21.9 (C-14),24.0 (C-15),175.4 (C-1′),109.4 (C-2′),174.9 (C-3′),109.1 (C-4′),173.6 (C-5′),109.1 (C-6′),194.0 (C-7′),193.7 (C-8′),43.5 (C-9′),38.7 (C-10′),30.0 (C-11′),25.2 (C-12′),23.4 (C-13′)。上述波谱数据与文献报道基本一致[9]，故鉴定化合物3为eucarobustol G。

化合物4：浅黄色固体。ESI-MSm/z: [M＋H]+469，分子式为C28H36O6。1H-NMR (400 MHz,pyridine-d5)δ: 10.28 (1H,s,CHO-7′),10.08 (1H,s,CHO-8′),5.02 (1H,s,H-14b),5.00 (1H,s,H-14a),4.83 (1H,s,H-12b),4.80 (1H,s,H-12a),4.25 (1H,d,J= 11.4 Hz,H-5),3.52 (1H,br d,J= 12.2 Hz,H-7),2.82 (1H,m,H-9a),2.81 (1H,dd,J= 12.1,2.0 Hz,H-6),2.55 (1H,dd,J= 6.8,2.0 Hz,H-9′),2.39 (1H,m,H-2b),2.27 (1H,brd,J= 13.3 Hz,H-9b),2.10 (1H,m,H-2a),1.98 (1H,m,H-8a),1.91 (1H,m,H-3b),1.85 (3H,s,H-13),1.73 (1H,m,H-11′),1.68 (1H,m,H-8b),1.49 (1H,m,H-10′b),1.29 (1H,m,H-3a),1.21 (1H,m,H-10′a),1.09 (3H,d,J= 6.8 Hz,H-13′),1.00 (3H,s,H-15),0.97 (3H,d,J= 6.9 Hz,H-12′)；13C-NMR (100 MHz,pyridine-d5)δ: 73.1 (C-1),33.3 (C-2),30.5 (C-3),36.5 (C-4),76.2 (C-5),46.3 (C-6),41.0 (C-7),24.7 (C-8),33.1 (C-9),148.1 (C-10),149.0 (C-11),107.7 (C-12),23.9 (C-13),110.2 (C-14),20.1 (C-15),169.2 (C-1′),104.4 (C-2′),168.0 (C-3′),104.5(C-4′),163.1 (C-5′),108.0 (C-6′),191.9 (C-7′),195.5 (C-8′),38.4 (C-9′),43.6 (C-10′),29.1 (C-11′),22.9 (C-12′),21.4 (C-13′)。上述波谱数据与文献报道基本一致[10]，故鉴定化合物4为 eucalyptal A。

化合物5：黄色粉末。ESI-MSm/z: 469 [M＋H]+，确定其分子式为C28H36O6。1H-NMR (400 MHz,pyridine-d5)δ: 10.32 (1H,s,7′-CHO),9.92 (1H,s,8′-CHO),5.12 (1H,s,H-14b),5.10 (1H,s,H-14a),4.27 (1H,d,J= 11.6 Hz,H-5),3.56 (1H,d,J= 11.6 Hz,H-6),2.97 (1H,ddd,J= 13.6,13.6,4.7 Hz,H-9b),2.86 (1H,dd,J= 13.6,4.2 Hz,H-8a),2.72 (1H,dd,J= 7.5,2.9 Hz,H-9′),2.48 (2H,ddd,J= 14.0,3.1,3.1 Hz,H-2b),2.34 (1H,m,H-9a),2.31 (1H,m,H-8b),2.20 (2H,ddd,J= 14.0,14.0,3.1 Hz,H-2a),2.20 (2H,ddd,J= 14.0,13.1,3.1 Hz,H-3b),1.84 (3H,s,H-13),1.78 (3H,s,H-12),1.29 (1H,m,H-10′a),1.60 (1H,m,H-10′b),1.50 (2H,ddd,J= 13.3,3.1,3.1 Hz,H-3a),1.16 (3H,d,J= 6.4,H-13′),1.11 (3H,s,H-15),0.91 (3H,d,J= 6.4,H-12′)；13C-NMR (100 MHz,pyridine-d5)δ: 73.9 (C-1),33.0 (C-2),30.5 (C-3),36.0 (C-4),75.8 (C-5),48.5 (C-6),128.0 (C-7),26.4 (C-8),33.2 (C-9),149.6 (C-10),127.0 (C-11),20.9 (C-12),20.7 (C-13),109.3 (C-14),19.6 (C-15),169.3 (C-1′),104.5 (C-2′),168.0 (C-3′),104.5 (C-4′),163.1 (C-5′),108.0 (C-6′),191.9 (C-7′),192.3 (C-8′),38.4 (C-9′),43.3 (C-10′),28.9 (C-11′),23.9 (C-12′),22.6 (C-13′)。上述波谱数据与文献报道基本一致[10]，故鉴定化合物5为eucalyptal C。

化合物6：无定形粉末。ESI-MSm/z: 401 [MH]-，确定其分子式为C23H30O6。1H-NMR (400 MHz,pyridine-d5)δ: 10.17 (1H,m,CHO-9),10.05 (1H,m,CHO-8),5.87 (1H,d,J= 10.0 Hz,H-2′),5.66 (1H,d,J= 10.0 Hz,H-3′),2.83 (1H,dd,J= 10.0,4.0 Hz,H-7),2.22 (1H,s,H-6′),1.90 (1H,dd,J= 13.6,2.8 Hz,H-5′a),1.79 (1H,overlapped,H-11),1.79 (1H,overlapped,H-8′),1.73 (1H,dd,J= 13.6,4.4 Hz,H-10a),1.70 (1H,m,H-10b),1.54 (3H,s,H-7′),1.51 (3H,d,J= 6.4 Hz,H-10′),1.50 (1H,m,H-5′b),0.99 (3H,d,J= 5.2 Hz,H-12),0.98 (3H,d,J= 6.0 Hz,H-13),0.94 (3H,m,H-9′)；13C-NMR (100 MHz,pyridine-d5)δ: 104.8 (C-1),161.9 (C-2),104.2 (C-3),168.0 (C-4),104.3 (C-5),169.5 (C-6),35.6 (C-7),192.2 (C-8),191.8 (C-9),43.9 (C-10),26.4 (C-11),23.5 (C-12),21.6 (C-13),77.3 (C-1′),137.3 (C-2′),131.4 (C-3′),73.0 (C-4′),38.5 (C-5′),38.1 (C-6′),27.9 (C-7′),36.7 (C-8′),16.8 (C-9′),17.1 (C-10′)。上述波谱数据与文献报道基本一致[11]，故鉴定化合物6为eucalyptin C。

化合物7：无定形粉末。ESI-MSm/z: 401 [MH]-，确定其分子式为C23H30O6。1H-NMR (400 MHz,CDCl3)δ: 10.14 (1H,s,9-CHO),10.01 (1H,s,CHO-8),5.91 (1H,m,H-2′),5.81 (1H,m,H-3′),3.17 (1H,m,H-7),2.50 (1H,m,H-10a),2.09 (1H,m,H-6′),2.03 (1H,dd,J= 12.6,2.8 Hz,H-5′a),1.82 (1H,m,H-8′),1.67 (1H,m,H-11),1.40 (1H,m,H-10b),1.39 (3H,s,H-7′),1.32 (1H,m,H-5′b),1.02 (3H,d,J= 6.4 Hz,H-12),1.00 (3H,s,H-13),1.00 (3H,s,H-10′),0.99 (3H,d,J= 7.0 Hz,H-9′)；13C-NMR (100 MHz,CDCl3)δ: 103.2 (C-1),163.5 (C-2),104.3 (C-3),168.0 (C-4),104.3 (C-5),170.8 (C-6),29.1 (C-7),192.4 (C-8),191.9 (C-9),36.2 (C-10),25.2 (C-11),24.0 (C-12),21.0 (C-13),77.0 (C-1′),137.9 (C-2′),130.7 (C-3′),73.6 (C-4′),30.7 (C-5′),36.2 (C-6′),24.2 (C-7′),35.0 (C-8′),16.4 (C-9′),16.8 (C-10′)。上述波谱数据与文献报道基本一致[11]，故鉴定化合物7为 eucalyptin D。

化合物8：浅黄色固体。ESI-MSm/z: 487 [M＋H]+，确定其分子式为C29H42O6。1H-NMR (400 MHz,pyridine-d5)δ: 10.56 (1H,s,7′-CHO),10.51 (1H,s,8′-CHO),3.62 (1H,m,H-9′),3.18 (3H,s,H-OCH3),2.74 (1H,brt,J= 12.0 Hz,H-10′a),2.33 (1H,overlapped,H-1),1.57 (1H,overlapped,H-10′b),1.36 (3H,s,H-15),1.23 (3H,s,H-12),1.18 (3H,s,H-14),1.14 (3H,s,H-13),1.03 (3H,s,H-12′),1.02 (3H,s,H-13′)；13C-NMR (100 MHz,pyridine-d5)δ: 53.9 (C-1),24.8 (C-2),40.7 (C-3),50.3 (C-4),49.5 (C-5),30.1 (C-6),26.7 (C-7),20.9 (C-8),38.0 (C-9),80.0 (C-10),19.9 (C-11),17.7 (C-12),29.5(C-13),19.0 (C-14),18.5 (C-15),171.2(C-1′),106.7 (C-2′),171.4(C-3′),106.8(C-4′),171.4(C-5′),108.8(C-6′),192.0(C-7′),192.2 (C-8′),41.6 (C-9′),36.4 (C-10′),27.9 (C-11′),22.2 (C-12′),25.4 (C-13′),48.5 (C-OCH3)。上述波谱数据与文献报道基本一致[11]，故鉴定化合物8为eucalyptin E。

化合物9：无定形粉末。ESI-MSm/z: 453 [MH]-，确定其分子式为C28H38O5。1H-NMR (400 MHz,pyridine-d5)δ: 10.55 (1H,s,H-13′),5.46 (1H,s,H-3),3.22 (1H,d,J= 1.6 Hz,H-8′a),3.20 (1H,d,J= 1.6 Hz,H-8′b),3.02 (1H,dd,J= 13.2,3.2 Hz,H-12′a),2.79 (1H,dd,J= 13.2,10.8 Hz,H-12′b),2.42 (1H,m,H-9′),1.88 (1H,m,H-8a),1.82 (1H,m,H-9a),1.69 (3H,s,H-15),1.46 (1H,m,H-8b),1.07 (1H,d,J= 3.6 Hz,H-9b),1.03 (3H,overlapped,H-10′),1.01 (3H,d,J= 2.0 Hz,H-12),1.00 (3H,overlapped,H-11′),0.93 (3H,s,H-13),0.87 (3H,s,H-14),0.54 (1H,m,H-7),0.46 (1H,m,H-6)；13C-NMR (100 MHz,pyridine-d5)δ: 43.9 (C-1),29.3 (C-2),121.5 (C-3),135.5 (C-4),44.6 (C-5),24.0 (C-6),19.8 (C-7),16.2 (C-8),34.5 (C-9),35.5 (C-10),18.5 (C-11),28.9 (C-12),16.1 (C-13),13.0 (C-14),21.7 (C-15),170.5 (C-1′),107.9(C-2′),173.0 (C-3′),106.9 (C-4′),167.6 (C-5′),105.8 (C-6′),206.8 (C-7′),23.1 (C-8′),25.8 (C-9′),23.4 (C-10′),23.4 (C-11′),23.1 (C-12′),193.2 (C-13′)。上述波谱数据与文献值基本一致[12]，故鉴定化合物9为eucalrobusone A。

化合物10：白色粉末。ESI-MSm/z: 453 [MH]-，确定其分子式为C28H38O5。1H-NMR (400 MHz,pyridine-d5)δ: 10.52 (1H,s,CHO-13′),5.36 (1H,d,J= 4.4 Hz,H-6),4.89 (1H,s,H-12b),4.84 (1H,s,H-12a),3.15 (2H,d,J= 6.4 Hz,H-8′),3.03 (1H,dd,J= 13.2,2.0 Hz,H-12′a),2.79 (1H,dd,J= 13.2,10.8 Hz,H-12′b),2.48 (1H,m,H-4),2.40 (1H,m,H-9′),1.88 (1H,overlapped,H-9),1.85 (1H,overlapped,H-2),1.81 (1H,overlapped,H-1),1.74 (3H,s,H-13),1.65 (1H,m,H-8),1.54 (1H,m,H-3),1.16 (3H,s,H-15),1.13 (3H,s,H-14),1.00 (3H,s,H-11′),0.98 (3H,s,H-10′)；13C-NMR (100 MHz,pyridine-d5)δ: 49.8 (C-1),23.4 (C-2),34.0 (C-3),40.0 (C-4),149.8 (C-5),124.2 (C-6),42.3 (C-7),23.3 (C-8),34.9 (C-9),39.1 (C-10),148.8 (C-11),112.4 (C-12),22.7 (C-13),22.1 (C-14),23.0 (C-15),170.1 (C-1′),108.5 (C-2′),172.8 (C-3′),106.8 (C-4′),167.4 (C-5′),105.6 (C-6′),206.8 (C-7′),53.4 (C-8′),25.7 (C-9′),23.4 (C-10′),23.4 (C-11′),23.6 (C-12′),193.2 (C-13′)。上述波谱数据与文献报道基本一致[12]，故鉴定化合物10为eucalrobusone C。

化合物11：白色粉末。ESI-MSm/z: [M－H]-383，确定其分子式为C23H28O5。1H-NMR (400 MHz,pyridine-d5)δ: 10.54 (1H,s,13′-CHO),7.21 (1H,d,J= 12.0 Hz,H-2),7.05 (1H,d,J= 7.6 Hz,H-5),7.00 (1H,dd,J= 7.6,1.2 Hz,H-4),4.13 (2H,s,H-12),3.14 (1H,d,J= 6.4 Hz ,H-8′),2.71 (1H,m,H-7),2.38 (1H,m,H-9′),2.19 (3H,s,H-10),1.08 (3H,s,H-8),1.07 (3H,s,H-9),1.00 (3H,s,H-10′),0.99 (3H,s,H-11′)；13C-NMR (100 MHz,pyridine-d5)δ: 138.9 (C-1),126.1 (C-2),147.2 (C-3),124.1 (C-4),130.7 (C-5),134.5 (C-6),34.5 (C-7),24.7 (C-8),24.7 (C-9),19.6 (C-10),171.0 (C-1′),106.0 (C-2′),173.1 (C-3′),106.0 (C-4′),168.5 (C-5′),105.2 (C-6′),206.7 (C-7′),53.2 (C-8′),25.8 (C-9′),23.4 (C-10′),23.4 (C-11′),26.7 (C-12′),193.4 (C-13′)。上述波谱数据与文献报道基本一致[12]，故鉴定化合物11为 eucalrobusone F。

化合物12：白色粉末。ESI-MSm/z: [M－H]-467，确定其分子式为C28H36O6。1H-NMR (400 MHz,pyridine-d5)δ: 10.33 (1H,s,CHO-13′),10.30 (1H,s,CHO-12′),4.73 (1H,d,J= 4.0 Hz,H-2),3.50 (1H,d,J= 11.2 Hz,H-7′),2.34 (2H,m,H-3),1.95 (1H,d,J= 11.2 Hz,H-5),1.31 (3H,s,H-15),1.27 (3H,s,H-14),1.03 (3H,s,H-12),1.00 (3H,s,H-13),0.98 (3H,d,J= 6.2 Hz,H-10′),0.87 (3H,d,J= 6.0 Hz,H-11′),0.56 (1H,m,H-6),0.47 (1H,m,H-7)；13C-NMR (100 MHz,pyridine-d5)δ: 75.8 (C-1),91.3 (C-2),39.9 (C-3),44.5 (C-4),47.4 (C-5),23.5 (C-6),26.1 (C-7),22.1 (C-8),32.4 (C-9),64.1 (C-10),20.7 (C-11),29.0 (C-12),16.4 (C-13),23.5 (C-14),23.9 (C-15),173.8 (C-1′),116.1 (C-2′),170.8 (C-3′),107.8 (C-4′),170.8 (C-5′),105.9 (C-6′),44.6 (C-7′),42.4 (C-8′),26.8 (C-9′),21.8 (C-10′),25.1 (C-11′),194.5 (C-12′),193.0 (C-13′)。上述波谱数据与文献报道基本一致[13]，故鉴定化合物12为 eucalrobusone U。

化合物13：无色粉末。ESI-MSm/z: 487 [M＋H]+，分子式为C28H38O7。1H-NMR (400 MHz,pyridine-d5)δ: 10.50 (1H,s,CHO-8′),10.48 (1H,s,CHO-7′),3.51 (H,dd,J= 11.8,4.0 Hz,H-9′),2.71 (1H,dd,J= 13.4,4.0 Hz,H-10′a),2.68 (1H,m,H-9b),2.65 (1H,m,H-2b),2.54 (1H,dd,J= 9.3,7.0 Hz,H-9a),2.50 (1H,d,J= 7.0 Hz,H-5),2.37 (1H,m,H-2a),2.30 (1H,m,H-8a),2.17 (1H,m,H-3b),2.05 (3H,s,H-14),1.78 (1H,dd,J= 9.3,7.0 Hz,H-8b),1.75 (1H,dd,J= 10.4,3.5 Hz,H-3a),1.55 (1H,m,H-11′),1.40 (1H,dd,J= 13.4,4.1 Hz,H-10′b),1.34 (3H,s,H-15),1.13 (3H,s,H-12),1.11 (3H,s,H-13),0.89 (3H,d,J= 6.6 Hz,H-13′),0.86 (3H,d,J= 6.5 Hz,H-12′),0.58 (1H,d,J= 7.2 Hz,H-7)；13C-NMR (100 MHz,pyridine-d5)δ: 221.0 (C-1),36.4 (C-2),32.9 (C-3),48.1 (C-4),21.2 (C-5),27.9 (C-6),25.2 (C-7),44.9 (C-8),55.3 (C-9),208.3 (C-10),18.3 (C-11),16.6 (C-12),29.4 (C-13),29.6 (C-14),22.1 (C-15),173.1 (C-1′),107.7 (C-2′),174.1 (C-3′),107.6 (C-4′),173.1 (C-5′),105.9 (C-6′),192.3 (C-7′),192.4 (C-8′),40.2 (C-9′),36.8 (C-10′),27.9 (C-11′),20.2 (C-12′),24.9 (C-13′)。上述波谱数据与文献报道基本一致[14]，故鉴定化合物13为eucalyptone。

化合物14：无色油状物。ESI-MSm/z: 387 [M＋H]+，分子式为C23H30O5。1H-NMR (400 MHz,CDCl3)δ: 9.93 (1H,s,CHO-8′),9.89 (1H,s,CHO-7′),5.79 (1H,dd,J= 10.1,4.1 Hz,H-1),5.45 (1H,dd,J= 9.8,1.9 Hz,H-6),2.70 (1H,m,H-9′),2.00 (1H,m,H-2),1.52 (3H,s,H-10)；13C-NMR (100 MHz,CDCl3)δ: 134.1 (C-1),38.6 (C-2),30.1 (C-3),38.0 (C-4),77.1 (C-5),129.1 (C-6),30.9 (C-7),20.0 (C-8),19.9 (C-9),27.5 (C-10),106.1 (C-1′),168.7 (C-2′),103.6 (C-3′),166.2 (C-4′),103.6 (C-5′),161.4 (C-6′),190.3 (C-7′),191.0 (C-8′),28.3 (C-9′),43.9 (C-10′),25.2 (C-11′),22.9 (C-12′),20.4 (C-13′)。上述波谱数据与文献报道基本一致[15]，故鉴定化合物14为euglobal-Ia1。

化合物15：无色油状物。ESI-MSm/z: 387 [M＋H]+，分子式为C23H30O5。1H-NMR (400 MHz,CDCl3)δ: 10.17 (1H,s,8′-CHO),10.06 (1H,s,7′-CHO),5.96 (1H,dd,J= 10.0,3.5 Hz,H-1),5.83 (1H,dd,J= 10.0,1.5 Hz,H-6),3.17 (1H,m,H-9′),2.04 (1H,m,H-2),1.80 (1H,m,H-10′b),1.57 (1H,dd,J= 9.4,11.4 Hz,H-10′a),1.40 (3H,s,H-10)；13C-NMR (100 MHz,CDCl3)δ: 134.3 (C-1),40.7 (C-2),22.4 (C-3),35.9 (C-4),78.0 (C-5),130.5 (C-6),31.9 (C-7),20.9 (C-8),20.6 (C-9),25.7 (C-10),104.9 (C-1′),169.8 (C-2′),103.9 (C-3′),167.8 (C-4′),104.4 (C-5′),163.3 (C-6′),191.4 (C-7′),192.1 (C-8′),29.7 (C-9′),36.8 (C-10′),25.3 (C-11′),23.5 (C-12′),21.8 (C-13′)。上述波谱数据与文献报道一致[16]，故化合物15鉴定为euglobal-Ia2。

4 细胞毒活性研究

采用MTT法[17]测定细胞毒活性。将培养好的第6代的MCF-7细胞以5×104个/孔的密度接种于96孔板，每孔200 μL，置于培养箱中过夜。然后3孔为1组，加入终浓度为20 μmol/L的待测单体化合物，对照组加入等量含DMSO的培养基，同时设置顺铂为阳性对照组，37 ℃、5%CO2培养箱中培养24 h。取出96孔板，向孔内加入提前放于室温下解冻的MTT溶液（5 mg/mL，20 μL/孔），37 ℃培养4 h后，弃上清液，每孔加DMSO 150 μl，震荡10 min后于1 h内在酶联免疫检测仪上测其波长为490 nm的吸光度（A）值，重复测定3次，按照公式计算相应的抑制率，见表1。