3.1 Isolation and structure identification of CPDs
Eventually, five novel Citrus peels (Chenpi) phenolic derivatives (CPDs) were isolated in a cell model of human intestinal epithelial (Caco-2) cells under BaP-exposure by a bio-assay guided method, named (3,4,5-trimethoxybenzoyl)valylphenylalanine (CPD-1), methyl (3,4,5-trimethoxybenzoyl)valylphenylalaninate (CPD-2), methyl (3,4,5-trimethoxybenzoyl)valylprolinate (CPD-3), methyl (3,4,5-trimethoxybenzoyl)valylvalinate (CPD-4), methyl 3-methyl-2-(3-methyl-2-(3,4,5-trimethoxybenzamido)butanamido)pentanoate (CPD-5). Furthermore, five known CPDs were also isolated and structure identified, named 1-(2,4-dihydroxy-6-methoxy-5-(1-methoxy-3-methylbutyl)-3-(3-methylbut-2-en-1-yl)phenyl)ethan-1-one (CPD-6), 1-(3-acetyl-2,6-dihydroxy-4-methoxy-5-(3-methylbut-2-en-1-yl)phenyl)-3-methylbutan-2-one (CPD-7), 1-(2,4-dihydroxy-6-methoxy-3-(3-methylbut-2-en-1-yl)phenyl)ethan-1-one (CPD-8), 2-butyryl-6-(3-butyryl-2,4-dihydroxy-6-methoxybenzyl)-3,5-dihydroxy-4,4-dimethylcyclohexa-2,5-dien-1-one (CPD-9), 2-acetyl-6-(3-butyryl-2,4-dihydroxy-6-methoxybenzyl)-3,5-dihydroxy-4,4-dimethylcyclohexa-2,5-dien-1-one (CPD-10). Their structures (Figure 1) were identified by 1H-NMR, 13C-NMR, and ESI-MS analyses. The 1H-NMR, 13C-NMR, and ESI-MS data of CPD-1~5 were list as follows:
CPD-1: White powder, 1H NMR (500 MHz, DMSO) δ: 0.68 (m, 6H, 2-CH3), 2.89 (m, 1H, -CH-), 3.07 (m, 2H, -CH2-), 3.71 (s, 3H, -OCH3), 3.83 (s, 6H, 2-OCH3), 4.31 (m, 1H, -CH-), 4.42 (m, 1H, -CH-), 7.19 (m, 7H, -CH-), 8.30 (m, 2H, 2-NH-); 13C NMR (125 MHz, DMSO) δ: 13C NMR (125 MHz, DMSO) δ 172.94, 172.75, 171.10, 152.48, 152.47, 139.99, 137.55, 129.54, 129.18, 129.14, 128.09, 128.04, 126.33, 105.17, 60.09, 58.97, 57.79, 56.07, 53.46, 30.18, 19.29, 18.98. ESI-MS: m/z 483.1 [M+Na]+, C24H30N2O7.
CPD-2: White powder, 1H NMR (600 MHz, DMSO) δ: 0.69 (m, 3H, -CH3), 0.90 (m, 3H, -CH3), 3.04 (m, 2H, -CH2-), 3.55 (s, 3H, -OCH3), 3.70 (s, 3H, -OCH3), 3.83 (s, 6H, 2-OCH3), 4.35 (m, 1H, -CH-), 4.48 (m, 1H, -CH-), 7.21 (m, 7H, 7-CH-), 8.18 (dd, 1H, -NH-), 8.51 (t, 1H, -NH-); 13C NMR (150 MHz, DMSO) δ: 171.78, 171.14, 165.60, 152.49, 152.47, 140.07, 140.02, 137.17, 137.08, 129.15, 129.07, 128.19, 128.15, 126.50, 126.48, 105.16, 105.15, 60.10, 58.68, 56.07, 56.05, 53.51, 51.71, 36.58, 30.34, 18.94, 18.58. ESI-MS: m/z 473.1 [M+H]+, 495.0 [M+Na]+, C25H32N2O7.
CPD-3: White powder, 1H NMR (600 MHz, DMSO) δ: 0.86 (m, 3H, -CH3), 0.91 (m, 3H, -CH3), 1.92 (m, 3H, -CH3), 2.16 (m, 2H, 2-CH-), 3.41 (m, 2H, -CH2-), 3.58 (s, 3H, -OCH3), 3.71 (s, 3H, -OCH3), 3.82 (s, 3H, -OCH3), 4.31 (m, 1H, -CH-), 4.58 (m, 1H, -CH-), 7.28 (m, 2H, 2-CH-), 8.54 (d, 1H, -NH-); 13C NMR (150 MHz, DMSO) δ: 172.26, 169.94, 152.52, 140.08, 128.98, 105.22, 60.08, 58.37, 56.05, 51.65, 46.87, 29.65, 28.68, 24.34, 19.16, 18.89. ESI-MS: m/z 423.7 [M+H]+, C21H30N2O7.
CPD-4: White powder, 1H NMR (600 MHz, DMSO) δ: 0.92 (m, 12H, 4-CH3), 2.06 (m, 1H, -CH-), 2.13 (m, 1H, -CH-), 3.60 (s, 3H, -OCH3), 3.69 (s, 3H, -OCH3), 3.84 (s, 6H, 2-OCH3), 4.14 (dt, 1H, -CH-), 4.41 (dt, 1H, -CH-), 7.21 (m, 2H, 2-CH-), 8.34 (dd, 2H, 2-NH-); 13C NMR (150 MHz, DMSO) δ: 171.84, 171.63, 165.80, 152.51, 140.03, 129.50, 105.17, 60.10, 58.86, 57.65, 56.09, 51.69, 30.50, 29.87, 19.35, 19.07, 18.91, 18.34. ESI-MS: m/z 447.1 [M+Na]+, C21H32N2O7.
CPD-5: White powder, 1H NMR (600 MHz, DMSO) δ: 0.92 (m, 12H, 4-CH3), 1.38 (m, 2H, -CH2-), 1.81 (m, 1H, -CH-), 2.11 (m, 1H, -CH-), 3.61 (s, 3H, -OCH3), 3.69 (s, 3H, -OCH3), 3.84 (s, 6H, -OCH3), 4.17 (dt, 1H, -CH-), 4.41 (dt, 1H, -CH-), 7.21 (m, 2H, 2-CH-), 8.28 (dd, 2H, 2-NH-); 13C NMR (150 MHz, DMSO) δ: 171.79, 171.43, 165.75, 152.51, 140.02, 129.50, 105.11, 60.08, 58.64, 56.57, 56.05, 51.65, 36.27, 30.55, 24.91, 19.30, 18.80, 11.19, 10.94. ESI-MS: m/z 439.2 [M+H]+, 461.2 [M+Na]+, C22H34N2O7.
After the ten CPDs were isolated, the cytotoxicity in Caco-2 cells was first evaluated (24, 48 and 72 h). The results showed that all the CPDs performed no cytotoxicity in Caco-2 cells up to 1.0 mM (72 h).
We then detected the protective activity of the CPDs against BaP-induced Caco-2 cells cell viability decline, and the results were list in Table 1. The BaP-exposure significantly decreased the cell viability. When compared to the control group, the BaP-exposure decreased the cell viability by 18.4% (p < 0.05), which indicated that the BaP-exposure induced fatal damages to the Caco-2 cells. Interestingly, BaP-exposure induced cell viability decrease was attenuated by most of the CPDs. Among them, the CPD-1, CPD-2 and CPD-3 displayed better protective activity than the resveratrol (positive control). Furthermore, the CPD-2 mostly promoted the viability of Caco-2 cells.
Table 1
CPDs inhibit BaP-induced Caco-2 cell viability decline
Group
|
Cell viability (100% Control)
|
Control
|
100 ± 4.42
|
BaP
|
81.6 ± 3.55##
|
CPD-1
|
93.2 ± 3.20*
|
CPD-2
|
95.7 ± 3.03**
|
CPD-3
|
89.5 ± 3.26*
|
CPD-4
|
86.1 ± 3.10
|
CPD-5
|
84.4 ± 3.23
|
CPD-6
|
85.4 ± 2.98
|
CPD-7
|
86.3 ± 3.12
|
CPD-8
|
84.2 ± 3.08
|
CPD-9
|
88.4 ± 3.13
|
CPD-10
|
88.3 ± 3.25
|
Resveratrol
|
89.8 ± 3.17*
|
The Caco-2 cells (1×104 cells/mL) were seeded in 96-well plates for 12 h. Then, the Caco-2 cells were treated with 1.0 µmol/L BaP or PBS. After incubation for 12 h, the Caco-2 cells were collected and washed with cold PBS for twice. Then, the Caco-2 cells were treated with the CPDs (10 µmol/L) or PBS for 24 h. Afterwards, the CCK-8 reagent was added to each well at 10 µL/mL final concentration and incubated at 37°C for another 4 h. Finally, cell viability of Caco-2 cells was determined with a microplate reader (Bio-Rad). Data represent means ± standard deviations (SD) for three independent experiments. (n=3, **p < 0.01, *p < 0.05, compared to the BaP group; #p < 0.05, ##p < 0.01, compared to the Control group). |
In current study, cytotoxicity of CPD-2 in Caco-2 cells was further explored. The results indicated that CPD-2 did not exhibit cycotoxicity up to 1.25 mM (72 h, Figure 2A). Furthermore, CPD-2 performed the protective activity against BaP-exposure induced cell viability decrease in a time- and dose-dependent manner (Figure 2B, 2C).