1 引言
2 结果与讨论
图2 (A)采用FBMN分析和SIRIUS注释对三种虎皮楠属植物生物碱部分进行化学成分系统性表征的技术流程、(B)利用Cytoscape构建的可视化分子网络、(C)目标DAs分子簇(簇一)[节点内字母(I~VIII)表示经SIRIUS预测注释的DAs, 节点旁数字表示前体离子质荷比(m/z)]及(D)经SIRIUS平台预测注释的已知DAs的分子结构以及其色谱保留时间和质谱峰Figure 2 (A) Technical workflow for systematic characterization of the chemical components in the alkaloid fractions of three Daphniphyllum species using FBMN analysis and SIRIUS annotation, (B) visualization of the molecular network constructed using Cytoscape, (C) selected molecular network clusters of DAs (Cluster 1) [The numbers (I~VIII) within the nodes indicate the DAs annotated by SIRIUS prediction, and the numbers adjacent to the nodes represent the precursor ion masses (m/z)], and (D) structures of known DAs predicted and annotated by the SIRIUS platform, along with their chromatographic retention times and mass peaks |
2.1 新化合物1~3的结构解析
表1 化合物1~3在CD3OD中的1H NMR (600 MHz)和13C NMR数据Table 1 1H (600 MHz) and 13C NMR data of compounds 1~3 in CD3OD |
| No. | 1 | 2 | 3 | |||||
|---|---|---|---|---|---|---|---|---|
| δH (J in Hz) | δCa | δH (J in Hz) | δCb | δH (J in Hz) | δCa | |||
| 1 | 216.5 | 3.84, s | 71.5 | 176.6 | ||||
| 2 | 2.45, m | 43.0 | 77.0 | 3.53, dt (10.3, 3.4) | 73.3 | |||
| 3 | a 2.64, br dd (16.1, 4.8) | 19.3 | α 1.94, m | 27.9 | α 1.94, ddt (16.6, 13.2, 3.6) | 39.6 | ||
| b 2.56, ddd (16.1, 4.7, 1.6) | β 1.71, m | β 1.60, ddt (16.6, 5.9, 3.2) | ||||||
| 4 | 4.01, dt (4.7, 2.0) | 88.2 | 2.10~2.16, m | 31.0 | 1.68~1.70, m | 44.7 | ||
| 5 | 53.4 | 42.4 | 43.2 | |||||
| 6 | 2.94, m | 48.0 | 2.49, overlapped | 38.7 | 2.40, overlapped | 41.9 | ||
| 7 | α 3.49, t (13.1) | 70.0 | a 3.40, dd (14.7, 1.8) | 57.9 | α 3.71, dd (13.8, 7.9) | 52.3 | ||
| β 3.26, dd (12.6, 6.0) | b 3.57, dd (14.7, 10.7) | β 3.49, dd (13.8, 10.7) | ||||||
| 8 | 67.4 | 42.4 | 58.4 | |||||
| 9 | 185.1 | 151.6 | 139.8 | |||||
| 10 | 3.62, br s | 38.1 | 2.99, m | 48.4 | 135.7 | |||
| 11 | α1.68, m | 24.7 | α 1.54, dd (13.4, 4.2) | 33.2 | 2.26-2.30, m | 25.4 | ||
| β 1.57, m | β 1.73, m | |||||||
| 12 | α1.79, m | 20.7 | α 1.42, m | 30.6 | α 1.66, overlapped | 26.4 | ||
| β 1.74, m | β 2.10, m | β 2.23, overlapped | ||||||
| 13 | α 2.32, dt (13.5, 8.9) | 40.5 | a 1.98, m | 29.4 | α 2.49, dd (13.0, 8.6) | 35.8 | ||
| β 3.02, m | b 2.22, overlapped | β 2.33, dd (13.0, 7.6) | ||||||
| 14 | 2.37~2.40, m | 25.4 | a 2.24, overlapped | 30.7 | 3.20, ddd (11.4, 8.6, 7.6) | 43.4 | ||
| b 2.44, m | ||||||||
| 15 | 153.3 | 5.84, t (2.5) | 131.5 | 3.30, overlapped | 54.2 | |||
| 16 | 205.7 | α 2.54, m | 30.2 | α 1.89, dt (11.8, 6.9) | 29.7 | |||
| β 2.23, overlapped | β 1.21, dtd (11.8, 10.5, 8.6) | |||||||
| 17 | α 2.99, dd (18.1, 6.1) | 49.6 | α 1.62, dd (12.5, 8.0) | 33.5 | α 2.49, overlapped | 43.0 | ||
| β 2.48, dd (18.1, 3.6) | β 2.10, m | β 2.22, overlapped | ||||||
| 18 | 2.59, m | 32.7 | 2.40, m | 44.2 | 2.13, dddq (10.3, 9.0, 8.1, 6.9) | 39.2 | ||
| 19 | α 3.16, dd (13.3, 10.8) | 67.1 | a 4.06, dd (12.8, 11.0) | 63.5 | α 4.32, dd (14.0, 8.1) | 54.1 | ||
| β 3.73, dd (13.3, 7.0) | b 2.78, dd (12.8, 8.9) | β 2.39, dd (14.0, 9.0) | ||||||
| 20 | 1.21, d (6.8) | 19.5 | 1.09, d (6.9) | 11.1 | 0.94, d (6.9) | 18.1 | ||
| 21 | 1.44, s | 22.8 | a 4.29, d (10.7) | 66.5 | 1.15, s | 25.4 | ||
| b 3.61, d (10.7) | ||||||||
| 22 | 175.9 | 177.3 | ||||||
| 23 | 3.62, s | 52.1 | 3.63, s | 51.8 | ||||
a Recorded at 125 MHz. b Recorded at 150 MHz. |
图5 计算与实验13C NMR化学位移之间的线性相关图Figure 5 Linear correlation plots between the calculated and experimental 13C NMR chemical shifts |
表2 (2R*)-2和(2S*)-2的DP4+概率结果Table 2 DP4+ probability for (2R*)-2 and (2S*)-2 |
| Possible isomer | (2R*)-2 | (2S*)-2 |
|---|---|---|
| DP4+ (H data) | 9.65% | 90.35% |
| DP4+ (C data) | 0.00% | 100.00% |
| DP4+ (all data) | 0.00% | 100.00% |
2.2 化合物生物活性测试结果
图6 (A)化合物1~13对红细胞溶血作用的96孔板观察结果、(B)化合物1~13的血液相容性评价、(C)化合物1~13对两种肿瘤细胞株的增殖抑制作用以及对P. falciparum Dd2的生长抑制作用及(D)活性化合物4与阳性对照对P. falciparum Dd2无性血期的剂量效应曲线[结果以平均值±SD表示(n=3)]Figure 6 (A) Visual inspection of RBC hemolysis induced by compounds 1~13 in 96‑well plates, (B) hemocompatibility evaluation of compounds 1~13, (C) cytotoxicity of compounds 1~13 against two tumor cell lines and their inhibitory activity against P. falciparum Dd2, and (D) dose‑response curve of compound 4 and positive controls against the asexual blood stages of P. falciparum Dd2 [The results were expressed as mean±SD (n=3)] |