Isolation and structural elucidation
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 recrystallization and various forms of column chromatography (CC) to afford compounds 1–26 (Figure 1).
Chemical structure of compound 4
Compound 4 was obtained as yellow solid with a molecular formula of C16H18O5 deduced from its HR-ESI-MS spectrum (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 the following: three aromatic proton signals [δH 7.96 (d, J = 7.1 Hz, 1H), 6.16 (d, J = 7.1 Hz, 1H), and 6.33 (d, J = 1.5 Hz, 1H)]; two methoxyl moieties [δH 3.94 (s, 3H) and 3.90 (s, 3H)]; two methyl [δH 1.68 (s, 3H) and 1.73 (s, 3H)]; and one methylene [δH 4.54 (dd, J =7.5, 1.5 Hz, 2H)]. The above nuclear magnetic resonance data are similar to those reported for compound 4′ in the literature [8-9].
Table 1 1H (600 MHz) and 13C (151 MHz) NMR data for compound 4 in CDCl3
Position
|
δH,m(J in Hz)
|
δC
|
HMBC
|
2
|
|
160.9
|
|
3
|
6.16, d, (7.1)
|
111.0
|
C-8a, C-2
|
4
|
7.96, d, (7.1)
|
138.8
|
C-5a, C-2, C-5
|
5
|
|
128.8
|
|
6
|
6.33, d (1.5)
|
91.3
|
C-8a, C-5, C-8, C-7
|
7
|
|
156.6
|
|
8
|
|
152.3
|
|
8a
|
|
103.9
|
|
5a
|
|
149.0
|
|
1′
|
4.54, dd (7.5, 1.5)
|
70.0
|
C-2′, C-5, C-3′
|
2′
|
5.57, d (1.5)
|
120.2
|
C-4′, C-5′
|
3′
|
|
139.0
|
|
4′
|
1.68, s
|
18.0
|
C-5′, C-2′, C-3′
|
5′
|
1.73, s
|
25.8
|
C-4′, C-2′, C-3′
|
7-OCH3
|
3.94, s
|
56.4
|
C-7
|
8-OCH3
|
3.90, s
|
56.4
|
C-8
|
A previous report [8] suggested the carbon signals of the C-8 and C-5 of compound 4' were slightly distinct with compound 4. Therefore, we speculate that the different carbon chemical shift at C-8 and C-5 may be caused by 3', 3'-dimethyl-2'-butenyloxy group positions. As illustrated in Figure 2, HMBC correlations of the protons H-1' (δH 4.54) with C-2′ (δC 120.2), C-3′ (δC 139.0), and C-5 (δC 128.8) indicated that the 3', 3'-dimethyl-2'-butenyloxy group of compound 4 is attached at the C-5 position. HMBC correlations of H-4 (δH 7.96) to C-5a (δC 149.0), C-2 (δC 160.9) and C-5 (δC 128.8); H-3 (δH 6.16) to C-8a (δC 103.9) and C-2 (δC 160.9) indicated that the lactone ring is close to C-8. Finally, the proton chemical shift for 7-OCH3 (δH 3.94, s), as based on HMBC data, correlates with the C-7 (δC 156.6), and the signal for 8-OCH3 (δH 3.90, s) correlates with the C-8 (δC 152.3). 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]. As the 13C-NMR data of compound 4 were not assigned in the literature, its 1D and 2D NMR data were analyzed in this study.
Chemical structure of compound 5
Compound 5 was isolated as a tawny oil. 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, and the IR spectrum showed absorptions for an α, β-unsaturated ester carbonyl (1731 cm−1) and an aromatic ring (1593 and 1430 cm−1). According to the 1H-NMR data in Table 2, there are three aromatic protons chemical shift [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 moiety [δH 3.65 (s, 2H)], and two methoxy [δH 3.84 (s, 3H) and 3.65 (s, 3H)]. The above nuclear magnetic resonance data indicated that compound 5 is consistent with 2-(5-methoxy-2-methyl-1H-indol-3-yl) methyl acetate, which has been previously reported in the literature [11].
Table 2 1H (600 MHz) and 13C (151 MHz) NMR data for compound 5 in CDCl3.
Position
|
δH,m(J in Hz)
|
δC
|
HMBC
|
2
|
|
132.8
|
|
3
|
|
128.9
|
|
4
|
7.04, m
|
111.1
|
C-5, C-3, C-7
|
5
|
|
154.1
|
|
6
|
6.75, dd (8.7, 2.4)
|
110.8
|
C-7, C-5, C-7a
|
7
|
6.98, d (8.7)
|
100.4
|
C-7a, C-5, C-6, C-4, C-4a
|
4a
|
|
104.1
|
|
7a
|
|
130.2
|
|
8
|
3.65, s
|
30.3
|
C-2, C-3, C-4a
|
9
|
|
172.8
|
|
10
|
2.28, s
|
11.7
|
C-2
|
5-OCH3
|
3.84, s
|
56.0
|
C-5
|
9-OCH3
|
3.65, s
|
52.0
|
C-9
|
Similar to compound 4, the 13C-NMR data for compound 5 was not reported in the previous literature, and the 1D and 2D NMR data were thus analysed. As depicted in Table 2, the coupling constant of the proton chemical shift at H-6 (δH 6.75) and H-7 (δH 6.98) is J = 8.7 Hz, suggesting that the two proton signals are ortho-coupled to the benzene ring. The three protons at δH 7.04 (m, 1H), δH 6.75 (dd, J = 8.7, 2.4 Hz, 1H), δH 6.98 (d, J = 8.7 Hz, 1H) correlated with carbons at δC 111.1, 110.8 and 100.4 in HSQC spetrum, respectively, indicated an aromatic ring. At the same time, the HMBC data (Figure 3) showed correlations of H-8 (δH 3.65) with C-2 (δC 132.8), C-3 (δC 128.9), and C-4a (δC 104.1), suggesting that the compound contains an indole moiety; and of H-10 (δH 2.28) with C-2 (δC 132.8), suggesting the presencen of a methyl acetate. Finally, the HMBC data revealed a correlation of 5-OCH3 (δH 3.84, s) with C-5 (δC 154.1) and of 9-OCH3 (δH 3.65, s) with C-9 (δC 172.8). These results indicated that the two -OCH3 groups are at C-5 and C-9. Compound 5 was thus named 2-(5-methoxy-2-methyl-1H-indol-3-yl) methyl acetate.
Chemical structure of compound 6
Compound 6 was isolated as a yellow oil. 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 and its 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 (Table 3) spectrum of compound 6 showed signals characteristic for two pairs of aromatic protons chemical shift [δH 7.73 (d, J = 8.7 Hz, 1H) and 7.50 (d, J = 8.7 Hz, 1H), 6.99 (d, J = 8.5 Hz, 1H) and 7.58 (d, J = 8.5 Hz, 1H)], two aromatic proton signals [δH 7.57 (s, 1H) and 7.12 (s, 1H)], two groups of methyl [δH 2.68 (s, 3H) and 1.21 (dd, J = 7.1 Hz, 3H)], three methylene moieties [δH 6.06 (s, 2H), 2.38 (s, 2H) and 4.17 (d, J = 7.1 Hz, 2H)], and two methoxy [δH 3.99 (s, 3H) and 3.95 (s, 3H)]. Compound 6 is a benzophenanthridine alkaloids based on the above nuclear magnetic resonance data. We found compound 6 to be consistent with 2'-(5, 6-dihydrochleletrythrine-6-yl) ethyl acetate, which has been previously reported in the literature [12].
Table 3 1H (600 MHz) and 13C (151 MHz) NMR data for compound 6 in CDCl3.
Position
|
δH,m(J in Hz)
|
δC
|
HMBC
|
1
|
7.12, s
|
104.3
|
C-2, C-12a, C-12
|
2
|
|
148.0
|
|
3
|
|
147.5
|
|
4
|
7.57, s
|
101.0
|
C-3, C-4b
|
4a
|
|
131.1
|
|
4b
|
|
139.3
|
|
6
|
5.02, m
|
55.1
|
C-4b, C-10a
|
6a
|
|
128.0
|
|
7
|
|
145.5
|
|
8
|
|
152.1
|
|
9
|
6.99, d (J= 8.5 Hz)
|
111.6
|
C-7, C-10a
|
10
|
7.58, d (J= 8.5 Hz)
|
118.8
|
C-8, C-10b, C-6a
|
10a
|
|
124.9
|
|
10b
|
|
123.8
|
|
11
|
7.73, d (J = 8.7 Hz)
|
119.8
|
C-4b, C-4a, C-10a
|
12
|
7.50, d (J = 8.7 Hz)
|
124.0
|
C-1, C-10b, C-12a
|
12a
|
|
127.5
|
|
N-CH3
|
2.68, s
|
42.9
|
C-6
|
7-OCH3
|
3.99, s
|
61.0
|
C-7
|
8-OCH3
|
3.95, s
|
55.8
|
C-8
|
-O-CH2-O-
|
6.06, s
|
101.0
|
|
1′
|
|
171.7
|
|
2′
|
2.38, s
|
39.2
|
C-1′, C-6
|
3′
|
4.17, d (J = 7.1 Hz)
|
60.3
|
|
4′
|
1.21, d (J = 7.1 Hz)
|
14.2
|
C-3′
|
The NMR data for compound 6 were assigned for the first time according to its 2D-NMR data. From the 1H-NMR data in Table 3, the coupling constant between the proton signals at H-11 (δH 7.73) and H-12 (δH 7.50) is J = 8.7 Hz, and that between H-9 (δH 6.99) and H-10 (δH 7.58) is J = 8.5 Hz, indicating that the two pairs of protons chemical shift are ortho-coupled to the phenyl ring. As depicted in Figure 4, HMBC data exhibited correlations of H-1 (δH 7.12) with C-2 (δC 148.0), C-12 (δC 124.0), and C-12a (δC 127.5) and of H-4 (δH 7.57) with C-3 (δC 147.5) and C-4b (δC 139.3), 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 55.1) also demonstrated 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 148.0), C-1′ (δC 171.7), and C-6 (δC 55.1) and of H-4′ (δH 1.21) with C-3′ (δC 60.3) suggested the presence of an ethyl acetate group. Finally, the HMBC correlations of 7-OCH3 (δH 3.99) with C-7 (δC 145.5) and of 8-OCH3 (δH 3.95) with C-8 (δC 152.1) indicated that the two -OCH3 groups are at C-7 and C-8.
Chemical Structure of compound 16
Compound 16 was obtained as tawny solid with a molecular formula of C13H11O4N deduced from its HR-ESI-MS spectrum (m/z 246.0760 [M + H]+). The UV profile of 16 revealed λ max values of 249, 201 and 316 nm, which are similar to those of quinoline [11]. The IR spectrum displayed absorption bands for an aromatic ring (1516 and 1443 cm−1) and an ether (1151 and 1046 cm−1). As indicated in Table 4, 1H-NMR detected two pairs of aromatic proton signals [δH 8.13 (d, J = 9.1 Hz, 1H) and 7.54 (d, J = 9.1 Hz, 1H), 7.15 (d, J = 2.7 Hz, 1H) and 7.80 (d, J = 2.7 Hz, 1H)], two methoxy moieties [δH 4.23 (s, 3H) and 4.27 (s, 3H)], and an active hydrogen chemical shift [δH 12.03 (s, 1H)]. Based on the above nuclear magnetic resonance data, compound 16 is consistent with 4-hydroxy-7, 8-dimethoxy-furoquinoline, which has been previously reported in the literature [14].
Table 4 1H (600 MHz) and 13C (151 MHz) NMR data for compound 16 in Pyridine-d5.
Position
|
δH,m(J in Hz)
|
δC
|
HMBC
|
2
|
|
164.5
|
|
3
|
|
101.6
|
|
4
|
|
142.3
|
|
4a
|
|
114.1
|
|
5
|
8.13, d (9.1)
|
118.8
|
C-4, C-8, C-8a
|
6
|
7.54, d (9.1)
|
117.3
|
C-7, C-8, C-4a
|
7
|
|
140.2
|
|
8
|
|
151.6
|
|
8a
|
|
157.4
|
|
3b
|
7.15, d (2.7)
|
105.3
|
C-2, C-3, C-4
|
2a
|
7.80, d (2.7)
|
142.9
|
C-2, C-3, C-3b
|
7-OCH3
|
4.23, s
|
61.1
|
C-7
|
8-OCH3
|
4.27, s
|
58.9
|
C-8
|
-OH
|
12.03, s
|
|
|
To clarify the structure of 16, we for the first time assigned its NMR data. The 1H-NMR data (Table 4), showed a coupling constant between the chemical shift at H-5 (δH 8.13) and H-6 (δH 7.54) is J = 9.1 Hz; these two proton signals are ortho-coupled to the phenyl ring. The HMBC data in Figure 5 illustrate the correlations of H-5 (δH 8.13) with C-4 (δC 142.3), C-8 (δC 151.6), and C-8a (δC 157.4) and of H-6 (δH 7.54) with C-6 (δC 117.3), C-8 (δC 151.6), and C-4a (δC 114.1), suggesting that compound 16 contains a quinoline ring. Similarly, the coupling constant between the chemical shift at H-3b (δH 7.15) and H-2a (δH 7.80) is J = 2.7 Hz, indicating that the protons are ortho-coupled to a furan ring. According to the HMBC data in Figure 5, correlations of H-3b (δH 7.15) with C-2 (δC 164.5), C-3 (δC 101.6), and C-4 (δC 142.3) and of H-2a (δH 7.80) with C-2 (δC 164.5), C-3 (δC 101.6), and C-3b (δC 105.3) suggest that this compound is a furan derivative. Finally, HMBC correlations of 7-OCH3 (δH 4.23) with C-7 (δC 140.2) and of 8-OCH3 (δH 4.27) with C-8 (δC 151.6) were observed. These results indicated that the two -OCH3 groups are located at C-7 and C-8. The above nuclear magnetic resonance data showed that compound 16 is consistent with 4-hydroxy-7, 8-dimethoxy-furoquinoline, which has been previously reported in the literature [14], though no 1D and 2D NMR data were reported. Herein, its NMR data of compound 16 were also assigned in the present study.
Overall, twenty-two compounds (compounds 5-26) were found to be alkaloids; the other four (compounds 1-4) were considered to be false-positive non-alkaloids based on the modified potassium caesium iodide test, as proven based on 1H-NMR and 13C-NMR spectra. In addition, by the comparison of NMR data with those described in the literature, the 26 compounds were identified as (+)-9′-O-transferuloyl-5, 5′-dimethoxylaricriresinol (1) [15], 8-(3′-oxobut-1′-en-1′-yl)-5, 7-dmethoxy-coumarin (2) [16], 5, 7, 8-trimethoxy-coumarin (3) [14], 5-(3′, 3′-dimethyl-2′-butenyloxy)-7, 8-dimethoxy-coumarin (4), 2-(5-methoxy-2-methyl-1H-indol-3-yl) methyl acetate (5), 2′-(5, 6-dihydrochleletrythrine-6-yl) ethyl acetate (6), 6-acetonyldi-hydrochelerythrine (7) [18], 6β-hydroxymethyldihydronitidine (8) [19], bocconoline (9) [20], zanthoxyline (10) [33], O-methylzanthoxyline (11) [21], rhoifoline B (12) [22], N-nornitidine (13) [23], nitidine (14) [24], chelerythrine (15) [25], 4-hydroxyl-7, 8-dimethoxy-furoquinoline (16), dictamnine (17) [26], γ-fagarine (18) [27], skimmianine (19) [13], robustine (20) [26], R-(+)-platydesmine (21) [28], 4-methoxyl-1-methyl-2-quinoline (22) [27], 4-methoxy-2-quinolone (23) [29], liriodenine (24) [30], aurantiamide acetate (25) [31], and 10-O-demethyl-12-O-methylarnottianamide (26) [32].
Biological activities of the isolated compounds
To analyse the effects of the 26 compounds on leukaemia cells (HEL cell lines), their IC50 values against HEL cells proliferation were determined by the CTG method, using adriamycin (IC50: 0.021 µM) as a positive control. As presented in Table 5, compound 14 (IC50: 3.59 µM) and compound 9 (IC50: 7.65 µM) showed the most potent inhibitory activities against HEL cells, compounds 15 (IC50: 15.52 µM) and 24 (IC50: 15.95 µM) exhibited moderate inhibitory activities against HEL cells. As the structures of compound 14 and compound 24 differ, different compounds of Z. nitidium may have inhibitory activity in HEL cells.
Table 5 Inhibitory activity of compounds 1-26 in HEL cell lines.
Compounds
|
IC50 (µM) ± SD
|
Compounds
|
IC50 (µM) ± SD
|
1
|
28.84 ±1.53
|
14
|
3.59 ± 0.82
|
2
|
22.43 ± 1.86
|
15
|
15.52 ± 0.26
|
3
|
>30
|
16
|
>30
|
4
|
>30
|
17
|
>30
|
5
|
>30
|
18
|
>30
|
6
|
>30
|
19
|
>30
|
7
|
>30
|
20
|
>30
|
8
|
>30
|
21
|
>30
|
9
|
7.65 ± 0.11
|
22
|
>30
|
10
|
24.94 ± 1.99
|
23
|
>30
|
11
|
>30
|
24
|
15.95 ± 2.33
|
12
|
>30
|
25
|
>30
|
13
|
>30
|
26
|
>30
|
DOX
|
0.021 ± 1.25
|
|
|
Compounds 14 and 24 induced cell cycle arrest
To confirm the effects of compounds 14 and 24 with different structures on the cell cycle, the cell cycle distribution of HEL cells was examined after treatment with the compounds for 36 h. As illustrated in Figure 6, significant S-transition arrest was observed in HEL cells treated with compound 14, which provided the most significant effect. Indeed, the fraction of cells in the S-phase was dose-dependently increased by treatment with 14, and the population of cells in S-phase was markedly increased to 52.04% in cells treated with 8 μM compared to 37.92% in untreated cells. Conversely, compound 24, with a different structure, had no obvious effect on the HEL cell cycle.
Compounds 14 and 24 induced apoptosis of HEL cells
To determine whether the antiproliferative activity of 14 and 24 is accompanied by enhanced leukaemia cell apoptosis, flow cytometry and an Annexin V-FITC apoptosis detection kit were used to detect apoptosis. Compared with untreated cells, cells treated with compounds 14 and 24 displayed significant dose-dependent increases, as shown in Figure 7. At the same time, compound 24 at 7.5 μM and 15.0 μM induced significant increases in apoptosis compared with the control group (DMSO). Compound 24 at concentrations of 7.5, 15 and 30 μM promoted apoptosis from 6.11% and 17.34% to 25.81% in a dose-dependent manner. Hence, compounds 14 and 24 caused obvious apoptosis in HEL cells in a concentration-dependent manner.