2.1 Isolation and Structural Elucidation
The dried roots and leaves (20 kg) of Z. nitidium were heated and refluxed in 95% EtOH. The resulting extract was concentrated and then partitioned between petroleum ether and chloroform. The extracts were further separated by solvent fractionation and various forms of column chromatography (CC) to afford compounds 1–26 (Figure 1).
2.1.1 Chemical Structure of Compound 4
Compound 4 was isolated as a yellow solid and gave a positive result with the improved caesium potassium iodide test. Its molecular formula was determined to be C16H18O5 based on its positive HR-ESI-MS data (m/z 291.1585 [M + H]+). The UV profile of 4 displayed the λ max values of 206, 263 and 323 nm, and its IR spectrum showed absorptions representing a lactone ring (1726 cm−1) and an aromatic ring (1502 and 1432 cm−1). The above data indicated that compound 4 contains a lactone ring. The 1H NMR data (Table 1) showed three aromatic proton signals at δH 7.96 (m, 1H), 6.16 (s, 1H), and 6.33 (d, J = 1.5 Hz, 1H); two methoxyl proton signals at δH 3.94 (s, 3H) and 3.90 (s, 3H); two methyl proton signals at δH 1.68 (s, 3H) and 1.73 (s, 3H); and one methylene signal at δH 4.54 (dd, J =7.5, 1.5 Hz, 2H). In addition, the 13C NMR and DEPT spectra of compound 4 showed the following groups: C × 7, CH × 4, CH2 × 1, OCH3 × 2, and CH3 × 2. The above nuclear magnetic resonance data are similar to the reported compound 4′ in the literature [8-9].
The 1D NMR signals of compound 4' and compound 4 were compared in Table 1. The proton signal at C-8 of compound 4' was the same as that of 4. As shown in Figure 2, the HMBC correlations of the protons at δH 4.54 (dd, J = 7.5, 1.5 Hz 2H) with C-2’ (δC 120.17), C-3’ (δC 139.03), and C-5 (δC 128.79) suggested that the 3', 3'-dimethyl-2'-butenyloxy group of compound 4 is attached at the C-5 position. The HMBC correlations of δH 7.96 with C-5a (δC 149.04), C-2 (δC 160.89), and C-5 (δC 128.79) and of δH 6.16 (s, 1H) with C-8a (δC 103.85) and C-2 (δC 160.89) indicate that the lactone ring is close to C-8. Finally, the proton signal for 7-OCH3 (δH 3.94, s), based on the HMBC data, is correlated with the signal for C-7 (δC 156.56), and the signal for 8-OCH3 (δH 3.90, s) is correlated with the signal for C-8 (δC 152.31). The two -OCH3 groups are at C-7 and C-8. The above nuclear magnetic resonance data indicated that compound 4 is consistent with 5-(3', 3'-dimethyl-2'-butenyloxy)-7, 8-methoxy-coumarin, which has been previously reported in the literature [10]. Due to describing compound 4 did not assign the NMR data, we assigned the NMR data of compound 4 for the first time.
2.1.2 Chemical Structure of Compound 5
Compound 5 was isolated as a tawny oil and gave a positive result in the improved caesium potassium iodide test, and it was therefore presumed to be an alkaloid. Its molecular formula was determined to be C13H15O3N based on its positive HR-ESI-MS data (m/z 234.1124 [M + H]+). The UV profile of 5 displayed the λ max values at 218 and 279 nm. The IR spectrum showed absorptions for an α, β-unsaturated ester carbonyl (1731 cm−1) and an aromatic ring (1593 and 1430 cm−1). The 1H NMR data in Table 2 showed that there are three aromatic protons with signals at δH 7.04 (m, 1H), 6.75 (dd, J = 8.7, 2.4 Hz, 1H), and 6.98 (d, J = 8.7 Hz, 1H), a methylene proton with a signal at δH 3.65 (s, 3H); and two methoxy protons with signals at δH 3.84 (s, 2H) and 3.65 (s, 3H). In addition, the 13C NMR and DEPT spectra of compound 5 indicated the presence of the following groups: C × 6, CH × 3, CH2 × 1, CH3 × 1 and OCH3 × 2. The above nuclear magnetic resonance data indicated that compound 5 is consistent with methyl 2-(5-methoxy-2-methyl-1H-indol-3-yl) acetate, which has been previously reported in the literature [11].
A previous study [11] describing compound 5 did not assign the NMR data. To further determine the structure of 5, we assigned the NMR data of compound 5 for the first time. As shown in the 1H NMR spectrum (Table 2), the coupling constant of the proton signals at δH 6.75 (dd, J = 8.7, 2.4 Hz, 1H) and δH 6.98 (d, J = 8.7 Hz, 1H) is J = 8.7 Hz, suggesting that the two proton signals are ortho-coupled on the benzene ring. The HSQC correlations between H-4 (δH 7.04) and C-4 (δC 111.14), between H-6 (δH 6.04) and C-6 (δC 110.83), and between H-7 (δH 6.98) and C-7 (δC 100.35) revealed that compound 5 contains an aromatic ring. At the same time, the HMBC data shown in Figure 3 show correlations of H-8 (δH 3.65) with C-2 (δC 172.85), C-3 (δC 128.86), and C-4a (δC 104.08) and of H-10 (δH 2.28) with C-4a (δC 104.08) and C-9 (δC 133.76), suggesting that the compound contains an indole moiety. Similarly, the HMBC (Figure 3) data showed correlations between H-8 (δH 3.65) and C-2 (δC 172.85), C-3 (δC 128.86), and C-4a (δC 104.08) and between H-10 (δH 2.28) and C-4a (δC 104.08) and C-9 (δC 133.76), suggesting the presence of a methyl acetate. Finally, the HMBC data showed correlation of 5-OCH3 (δH 3.84, s) with C-5 (δC 154.05) and of 9-OCH3 (δH 3.65, s) with C-9 (δC 133.76). These results indicate that the two -OCH3 groups are at C-5 and C-9. Compound 5 was thus named methyl 2-(5-methoxy-2-methyl-1H-indol-3-yl) acetate.
2.1.3 Chemical Structure of Compound 6
Compound 6 was isolated as a yellow oil, gave a positive result in the improved caesium potassium iodide test, and was therefore presumed to be an alkaloid. Its molecular formula was determined to be C25H25O6N based on its positive HR-ESI-MS data (m/z 436.1752 [M + H]+). The UV profile of 6 revealed λ max values of 201, 283 and 224 nm. The IR spectrum showed absorption bands for an α, β-unsaturated ester carbonyl (1736 cm−1) and an aromatic ring (1492 and 1463 cm−1). The 1H NMR data (Table 3) showed that there were two pairs of aromatic protons with signals at δH 7.73 (d, J = 8.7 Hz, 1H) and 7.50 (d, J = 8.7 Hz, 1H) and at 6.99 (d, J = 8.5 Hz, 1H) and 7.58 (d, J = 8.5 Hz, 1H); two aromatic protons with signals at δH 7.57 (s, 1H) and 7.12 (s, 1H); two groups of methyl protons with signals at δH 2.68 (s, 3H) and 1.21 (dd, J = 7.1 Hz, 3H); three groups of methylene protons with signals at δH 6.06 (s, 2H), 2.38 (s, 2H) and 4.17 (d, J = 7.1 Hz, 2H); and two groups of methoxy protons with signals at δH 3.99 (s, 3H) and 3.95 (s, 3H). In addition, the 13C NMR and DEPT spectra of compound 6 indicated the presence of the following groups: C × 11, CH × 7, CH2 × 3, CH3 × 2 and OCH3 × 2. The above nuclear magnetic resonance data indicated that compound 6 is a benzophenanthrene alkaloid. We found that compound 6 was consistent with ethyl 2’-(5, 6-dihydrochleletrythrine-6-yl) acetate, which has been previously reported in the literature [12].
The previous study [12] of compound 6 did not assign its NMR data. To clarify the structure of 6, we assigned the NMR data of 6 for the first time. From the 1H NMR data in Table 3, the coupling constant between the proton signals at δH 7.73 (d, J = 8.7 Hz, 1H) and 7.50 (d, J = 8.7 Hz, 1H) is J = 8.7 Hz, and that between δH 6.99 (d, J = 8.5 Hz, 1H) and 7.58 (d, J=8.5 Hz, 1H) is J = 8.5 Hz, indicating that the two pairs of proton signals are ortho-coupled on the phenyl ring. As shown in Figure 4, the HMBC data exhibited the correlations of H-1 (δH 7.12) with C-2 (δC 147.95), C-12 (δC 123.99), and C-12a (δC 127.53) and of H-4 (δH 7.57) with C-3 (δC 147.50) and C-4b (δC 139.30), indicating that compound 6 is a benzophenanthrene derivative. The direct HSQC (Figure S19, Supplementary Materials) correlations between H-6 (δH 4.95) and C-6 (δC 55.11) also revealed that compound 6 is a chelerythrine. Similarly, based on the HMBC (Figure 4), the correlations of H-2’ (δH 2.38) with C-2 (δC 172.85), C-1’ (δC 171.67), and C-6 (δC 55.11) and of H-4’ (δH 1.21) with C-3’ (δC 60.27) suggest the presence of an ethyl acetate group. Finally, the HMBC correlations of 7-OCH3 (δH 3.99, s) with C-7 (δC 145.50) and of 8-OCH3 (δH 3.95, s) with C-8 (δC 152.10) suggested that the two -OCH3 groups were at C-7 and C-8.
2.1.4 Chemical Structure of Compound 16
Compound 16 was isolated as a tawny solid, gave a positive result with the improved caesium potassium iodide test, and was therefore presumed to be an alkaloid. Its molecular formula was determined to be C13H11O4N based on its positive HR-ESI-MS data (m/z 246.0760 [M + H]+). The UV profile of 16 revealed the λ max values of 249, 201 and 316 nm, which are similar to those of quinoline [11]. The IR spectrum showed the absorption bands for an aromatic ring (1516 and 1443 cm−1) and an ether (1151 and 1046 cm−1). The 1H NMR data in Table 4 showed two pairs of aromatic proton signals at δH 8.13 (d, J = 9.1 Hz, 1H) and 7.54 (d, J = 9.1 Hz, 1H), and at 7.15 (d, J = 2.7 Hz, 1H) and 7.80 (d, J = 2.7 Hz, 1H), two methoxy proton signals at δH 4.23 (s, 3H) and 4.27 (s, 3H), and an active hydrogen signal at δH 12.03 (s, 1H). In addition, the 13C NMR and DEPT spectra of compound 16 indicated the presence of the following groups: C × 7, CH × 4 and OCH3 × 2. Based on the above nuclear magnetic resonance data, compound 16 was consistent with 4-hydroxy-7, 8-demethy-furoquinoline, which has been previously reported in the literature [14].
To clarify the structure of 16, we assigned the NMR data of compound 16 for the first time. Based on the 1H NMR data in Table 4, which showed a coupling constant between the proton signals at δH 8.13 (d, J = 9.1 Hz, 1H) and 7.54 (d, J = 9.1 Hz, 1H) of J = 9.1 Hz, these two proton signals are ortho-coupled on the phenyl ring. The HMBC data in Figure 5 showed the correlations of H-5 (δH 8.13) with C-4 (δC 142.30), C-8 (δC 151.59), and C-8a (δC 157.41) and of H-6 (δH 7.54) with C-6 (δC 117.32), C-8 (δC 151.59), and C-4a (δC 114.11), suggesting that compound 16 contains a quinoline ring. Similarly, the coupling constant between the proton signals at δH 7.15 (d, J = 2.7 Hz, 1H) and δH 7.80 (d, J = 2.7 Hz, 1H) is J = 2.7 Hz, indicating that the protons are ortho-coupled on a furan ring. In addition, from the HMBC data in Figure 5, the correlations of H-3b (δH 7.15) with C-2 (δC 164.48), C-3 (δC 101.61), and C-4 (δC 142.30) and of H-2a (δH 7.80) with C-2 (δC 164.48), C-3 (δC 101.61), and C-3b (δC 105.34) suggest that this compound is a furan derivative. Finally, HMBC correlations of 7-OCH3 (δH 4.23, s) with C-7 (δC 140.17) and of 8-OCH3 (δH 4.27, s) with C-8 (δC 151.59) were observed. These results indicated that the two -OCH3 groups were at C-7 and C-8.
By the comparison of their NMR data with those described in the literature, twenty-six compounds were identified as (+)-9’-O-transferuloyl-5, 5’-dimethoxylaricriresinol (1) [15], 8-(3’-oxobut-1’-en-1’-yl)-5, 7-trimethoxy-coumarin (2) [16], 5, 7, 8-trimethoxy-coumarin (3) [14], 5-(3', 3'-dimethyl-2'-butenyloxy)-7, 8-trimethoxy-coumarin (4), methyl 2-(5-methoxy-2-methyl-1H-indol-3-yl) acetate (5), ethyl 2’-(5, 6-dihydrochleletrythrine-6-yl) acetate (6), 6-acetonyldi-hydrochelerythrine (7) [18], 6β-hydroxymethyldihydronitidine (8) [19], bocconoline (9) [20], zanthoxyline (10) [21], O-methylzanthoxyline (11) [21], rhoifoline B (12) [22], N-nornitidine (13) [23], nitidine (14) [24], chelerythrine (15) [25], 4-hydroxyl-7, 8-demethyfuroquinoline (16), dictamnine (17) [26], γ-fagarine (18) [27], skimmianine (19) [13], robustine (20) [26], R-(+)-platydesmine (21) [28], 4-O-methyl-1-methyl-quinoline-2-one (22) [27], 4-methoxy-2-quinolone (23) [29], liriodenine (24) [30], aurantiamide acetate (25) [31], and 10-O-demethyl-12-O-methylarnottianamide (26) [32].
2.2 Biological Activities of the Isolated Compounds
To analysis the effects of 26 compounds for isolated from the roots and leaves of Z. nitidium against leukaemia cells (HEL cell lines), 26 compounds were tested of IC50 value against HEL by the CTG method, and Adriamycin was chosen as positive control (IC50: 0.021 µM). As shown in Table 5, the most potent compound 14 (IC50: 3.59 µM) and compound 9 (IC50: 7.65 µM) showed the similar inhibitory activity with the positive control (IC50: 0.021 µM), while these compounds 15 (IC50: 15.52 µM) and 24 (IC50: 15.95 µM) exhibited moderate inhibitory activities against HEL cells. In addition, compound 24 whose structure type is different from 14 also exhibited good inhibitory activity against HEL.
2.3 Compounds 14 and 24 Induced cell cycle arrest
To further confirm the effects of compounds 14 and 24 with different structures on cell cycle, the cell cycle of distribution of HEL cells was examined after treatment with compounds 14 and 24 for 36 h. As shown in Figure 6, significant S transition arrest was observed in HEL cells treated with compound 14, which was the most significant compound. The fraction of cells in the S phase was dose-dependently increased by the treatment with 14, and the population of cells in the S phase was markedly increased to 52.04 % in 8 μM 14-treated cells compared to 37.92 % in untreated cells. However, compound 24 with different structure type has no obvious effect on the cycle experiments against HEL cells.
2.4 Compounds 14 and 24 induced apoptosis of HEL cells
To determine whether the antiproliferative activity of 14 and 24 was accompanied by enhanced leukaemia cell apoptosis, cell apoptosis was detected by a flow cytometry assay after staining with an Annexin V-FITC apoptosis detection kit. As shown in Figure 7, Cells treated with compounds 14 and 24 displayed significant dose-denpendent increases in the percentage of Annexin-V-positive cells. Compound 14 from 1.86 % in the DMSO control to 13.99 % for 2.0 μM, 23.96 % for 4.0 μM and 35.98 % for 8.0 μM 14-treated cells. At the same time, compound 24 at 7.5 μM and 15.0 μM displayed significant increases in the percentage of Annexin-V-positive cells. Compound 24 (7.5, 15, 30 μM) can promote the apoptosis rate from 6.11%, 17.34%, 25.81% in a dose-dependent manner. Hence, these observations for the first time demonstrated that compounds 14 and 24 induced obvious apoptosis in leukaemia cells HEL in a concentration-dependent manner.