Supplementary Materials Identification of novel Nrf2 activators from Cinnamomum chartophyllum H. W. Li and their potential application of preventing oxidative insults in human lung epithelial cells Ming‐Xing Zhou1, Guo‐Hui Li2, Bin Sun3, You‐Wei Xu1, Ai‐Ling Li1, Yan‐Ru Li1, Dong‐Mei Ren1, Xiao‐Ning Wang1, Xue‐Sen Wen1, Hong‐Xiang Lou1, Tao Shen*,1 1
Key Lab of Chemical Biology (MOE), School of Pharmaceutical Sciences, Shandong University, Jinan, P. R. China;
2
Department of Pharmacy, Jinan Maternity and Child Care Hospital, Jinan, P. R. China
3
National Glycoengineering Research Center, Shandong University, Jinan 250012, PR China
* Corresponding author at: School of Pharmaceutical Sciences, Shandong University, 44 Wenhua Xi Road, Jinan 250012, P. R. China; Tel.: 86‐531‐88382028; fax: 86‐531‐88382548; E‐mail address:
[email protected] (T. Shen)
1. Extraction and isolation of constituents from Cinnamomum chartophyllum The air‐dried and powdered aerial parts (5.4 kg) of C.chartophyllum were extracted with 95% EtOH (10 L × 4). The solvent was removed under reduced pressure to yield an EtOH extract (300.4 g). The dried EtOH extract was suspended in water, partitioned successively with petroleum ether, EtOAc and n‐butanol and give three partitions. The petroleum ether soluble partition (15.7 g) was separated over silica gel column chromatography (CC) and eluted with a gradient of petroleum etherEtOAc (100:00:100) to yield twenty fractions (Frs.P1P20). Compounds 22 (12.3 mg), 29 (4.1 mg) and 30 (5.2 mg) were precipitated from Frs.P1, P6, and P7, respectively. Fr. P9 was subjected to silica gel CC using a gradient of petroleum etherEtOAc (100:080:20) to give 27 (5.7 mg) and 28 (4.3 mg). Fr. P10 was separated on a Sephadex LH‐20 column, eluting with CH2Cl2/MeOH (1:1) to furnish 26 (3.9 mg). Compounds 23 (9.3 mg) and 21 (20.9 mg) were precipitated from Frs. P11 and P14, respectively. Fr. P15 was chromatographed on Sephadex LH‐20 eluted with CH2Cl2/MeOH (1:1) to afford six subfractions (Frs. P15aP15f). Frs.P15d and P15e were purified by semi‐preparative HPLC 1
Supplementary Materials (MeOH/H2O=70:3085:15, 020 min, MeOH/H2O=85:15, 2050 min; 1.5 mL/min) to give 1 (3.8 mg, tR=7.8 min), 8 (2.6 mg, tR=13.5 min) and 3 (3.9 mg, tR=27.7 min). Separation of Fr. P16 using Sephadex LH‐20 CC with CH2Cl2/MeOH (1:1) as eluent produced eight subfractions (Frs.P16aP16h). Fr. P16e was purified by semi‐preparative HPLC (MeOH/H2O=95:5; 1.5 mL/min) to yield five subfractions (Frs.P16e1‐P16e5). Fr.P16e1 was further purified by semi‐preparative HPLC (MeOH/H2O=40:60; 1.0 mL/min) to afford 10 (2.4 mg, tR=16.9 min), 11 (2.8 mg, tR=18.1 min), and 17 (4.2 mg, tR=29.3 min). Similarly, Fr.P16e3 was further purified by semi‐preparative HPLC (MeOH/H2O=57:43, 1.0 mL/min) to afford 12 (2.0 mg, tR=54.3 min). 19 (1.6 mg, tR=31.9 min) was purified from Fr. P16f by semi‐preparative HPLC (MeOH/H2O=40:6080:20, 045 min; 1.0 mL/min). Fr. P18 was fractionated by Sephadex LH‐20 CC eluting with CH2Cl2/MeOH (1:1), to afford six subfractions (Frs. P18aP18f). Fr. P18e was subjected to semi‐preparative HPLC (MeOH/H2O= 20:8080:20, 070 min; 1.0 mL/min) to give 18 (1.0 mg, tR=39.2 min). The EtOAc‐soluble partition (33.8 g) was separated on a silica gel CC using a gradient of petroleum etherEtOAc (100:00:100) to afford nineteen fractions (Frs. E1E19). Compounds 24 (3.1 mg) and 25 (4.4 mg) were precipitated from Frs. E8 and E9, respectively. Fr. E13 was separated by a Sephadex LH‐20 CC eluting with CH2Cl2/MeOH (1:1) to afford nine subfractions (Frs.E13aE13i). Fr. E13e was purified by semi‐preparative HPLC (MeOH/H2O=50:5075:25, 030 min; 1.5 mL/min) to afford 9 (2.5 mg, tR=21.6 min). Fr. E13h was submitted to a Sephadex LH‐20 CC using CH2Cl2/MeOH (1:1) as eluent to yield six fractions (Frs. E13h1E13h6). Fr.E13h5 was subjected to semi‐preparative HPLC (MeOH/H2O= 50:50100:0, 085 min; 1.5 mL/min) to give 14 (10.3 mg, tR=14.1 min), 20 (2.0 mg, tR=21.5 min), 15 (17.8 mg, tR=22.6 min) and 4 (2.0 mg, tR=58.3 min).Fr. E14 was fractionated by CC on Sephadex LH‐20 and eluted with CH2Cl2/MeOH (1:1), affording ten subfractions (Frs.E14aE14j). Fr. E14f was separated by semi‐preparative HPLC (MeOH/H2O=40:60; 1.5 mL/min) to yield 16 (33.0 mg, tR=19.1 min). Fr. E14h was divided into seven subfractions (Frs. E147h1E147h7) by semi‐preparative HPLC (MeOH/H2O=40:6080:20, 070 min; 1.5 mL/min) to afford 13 (13.5 mg, tR=13.1 min), 7 (4.1 mg, tR=37.5 min). Fr. E14i was subjected to semi‐preparative HPLC (MeOH/H2O=10:90100:0, 050 min; 1.0 mL/min) to give 2 (1.5 mg, tR=20.7 min) and 5 (1.1 mg, tR=37.5 min). Fr. E14j was purified by semi‐preparative HPLC (MeOH/H2O=40:60100:0, 060 min; 1.0 mL/min) to yield 6 (2.6 mg, tR=21.5 min). 2
Supplementary Materials R2
O
O
O
OH
HO
OH
OH
O
HO
H
R3
O
O
2
O
H
OH 1
R1
OH O
O
3
4 R1=OCH3, R2=R3=H 5 R1=R3=OH, R2=H 6 R1=R2=R3=OH
OH OH HO
O
HO
O
O
O
OH OH O
OH 7
O
8
HO OH
10 6 -OH 11 6 -OH
9
12
OR2 O
O OH
O
OR1 13 R1=R2=H 14 R1=CH3, R2=H 15 R1=H, R2=CH2CH3
O
OH OH H H O
O
O
O
OH
16
18
17
O HO
HO
O H
H O
O H
O
OH O HO
O
21
20
19
O n OH 22
n
O OH
25 n=16 26 n=20 27 n=22 28 n=23
23 n=25 24 n=29
Fig. S1. Chemical structures of the purified constituents 3
n O 29 n=26 30 n=30
13
Supplementary Materials 2. Names of the purified constituents The structures of isolated constituents were identified to be 3S‐(+)‐9‐oxonerolidol (NLD, 1)[1], 3,3',4,4'‐tetrahydroxydiphenyl (THD, 2)[2], L‐sesamin (3)[3], genkwanin (4)[4], kaempferol (5)[5], quercetin (6)[6], 3',4',5,7‐tetrahydroxyflavanone (7)[7], 5α‐hydroxy‐2‐oxo‐p‐menth‐6(1)‐ene (8)[8], 8‐hydroxycarvotanacetone
(9)[9],
(4R,6R)‐6‐hydroxypiperitone
(10)[10],
(4S,6R)‐6‐hydroxypiperitone (11)[10], 2‐methyl‐6‐(p‐tolyl) heptane‐2,3‐diol (12)[11], protocatechuic acid (13)[12], isovanillic acid (14)[13], ethyl protocatechuate (15)[14], 1,3,5‐trimethoxybenzene (16)[15],
4‐hydroxy‐4,7‐dimethyl‐1‐tetralone
rel‐(3R,3'S,4R,4'S)‐3,3',4,4'‐tetrahydro‐6,6'‐dimethoxy[3,3'‐bi‐2H‐benzopyran]‐4,4'‐diol
(17)[16], (18)[17],
piperitol (19)[18], 5,7‐dihydroxychromone (20)[19], β‐sitosterol (21)[20], n‐dotriacontane (22)[21], n‐octacosanol (23)[22], n‐dotriacontanol (24)[23], n‐eicosanic acid (25)[24], lignoceric acid (26)[25], hexacosanoic acid (27)[26], heptacosanoic acid (28)[27], octacosyl palmitate (29)[28], and n‐ dotriacontanyl hexadecanoate (30)[29]. Their structures were established by comparison of their NMR and MS data with those reported in the literature. 3. Cytotoxicity of NLD and THD against human lung epithelial Beas‐2B cells
Fig. S2. No significant toxicity was observed for NLD ≤ 100 μM, and for THD ≤ 25 μM against human lung epithelial Beas‐2B cells. Cells were treated with different concentrations for 48 h, and then cell viability was determined by the MTT assay. Results are expressed as mean ± SD (n=3). *p