Chemical constituents from ethanol extract of Polyalthia rumphii branches and their cytotoxicity evaluation

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Chemical constituents from ethanol extract of Polyalthia rumphii branches and their cytotoxicity evaluation

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Tianshan Wang a,b,∗,1 , Xiaopeng Wu c,1 , Yanping Liu a,b , Youping Luo a,b , Xiang Li a , Yujie Zhou a , AiPing Yang a , Zhengmeng Yan a , Ling Ye a , Siwei Chen a , Jinshuai Fu a , Xianzhi Jiao a a

College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, China Key Laboratory of Tropical Medicinal Plant Chemistry (Hainan Normal University), Ministry of Education, Haikou, China c Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, Haikou, China b

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Article history: Received 12 July 2017 Accepted 20 March 2018 Available online xxx

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Keywords: Alkaloids Lignan Gossypol derivative Cytotoxic activity Polyalthia rumphii

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Introduction

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Twelve known compounds, including eight alkaloids, three lignans and one gossypol derivative, were isolated from the branches of Polyalthia rumphii (Blume ex Hensch.) Merr., Annonaceae. The chemical structures were determined by spectroscopic methods and comparison with literature data. All the isolates were evaluated the cytotoxicity against three human cancer cell lines: Hela, MCF-7 and A549, the results showed that partial of isolates displayed weak cytotoxicities with the IC50 values ranging from 25 to 40 ␮g/ml. © 2018 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Polyalthia rumphii (Blume ex Hensch.) Merr., Annonaceae, distributes widely in tropic and subtropic area (Editorial Board of FOC, 2011). Chinese used many species of this genus as 25 traditional medicinal plants to treat intractable diseases (Yuan 26 et al., 2011). Especially Li ethnic minority, located in Hainan 27 28 Island, used the extracts of P. rumphii for prevention of fever, 29 hypertension and inhibition of cancer cells (Yuan et al., 2011; 30 Machana et al., 2012). Previous phytochemical research reported 31 that alkaloids and lignans, isolated from P. rumphii, were with 32Q3 anticancer activities (Wang et al., 2012a,b, 2013). As part of 33 our ongoing purpose of screening the bioactive constituents 34 from P. rumphii, this present investigation described the iso35 lation, structure identification, and all isolates were evaluated 36 the cytotoxic activities against three human cancer cell lines 37 by MTT assay. By chromatographic methods, twelve know com38 pounds were isolated, the chemical structures of them were 39 mainly determined by physical and spectroscopic methods includ40 ing [␣]D , NMR and MS as well as by comparing with literature 41 data. 23

Materials and methods

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General

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NMR spectra were recorded on a Bruker AV-400, deuterated reagents were CDCl3 (TMS as internal standard) and CD3 OD (residual solvent as internal standard). ESI-MS was measured on a Bruker amaZon SL mass spectrometer, and HR-ESI-TOF-MS were measured on an AB Sciex TripleTOF 5600+ high-resolution mass spectrometer. Preparative HPLC were performed on a Waters 2545-2767-2489 HPLC system. The single-crystal was diffracted on an X-Ray Diffractometer of Bruker Smart Apex-II CCD. Plant material Fresh branches of P. rumphii (Blume ex Hensch.) Merr., Annonaceae, were collected in September 2013, from Bawang Mountain of Hainan Island, China, and authenticated by Prof. Qiongxin Zhong (Hainan Normal University). The voucher specimen (No. 201309XHAL) was deposited at our key laboratory. Extraction and isolation

∗ Corresponding author. E-mail: [email protected] (T. Wang). 1 These authors contributed equally to this manuscript.

The branches of P. rumphii were air-dried at room temperature, and then reduced to coarse powder by crushing machine. This coarse powder (6.78 kg) was extracted in July 2014, with 95% EtOH

https://doi.org/10.1016/j.bjp.2018.03.004 0102-695X/© 2018 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Wang, T., et al. Chemical constituents from ethanol extract of Polyalthia rumphii branches and their cytotoxicity evaluation. Revista Brasileira de Farmacognosia (2017), https://doi.org/10.1016/j.bjp.2018.03.004

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(15 l × 3) at room temperature, and the solvents were evaporated under reduced pressure to get crude EtOH gum, which was continuously suspended in 0.1% H2 SO4 (4 l). The liposoluble components were removed by PE (petroleum ether) from acidic solution, and the acidic aqueous solution was extracted by EtOAc (4 l × 3) to get part I (94.8 g), the remaining aqueous layer was alkalinized (pH 9–10) by ammonia and extracted with CHCl3 (4 l × 3) to get part II (48.1 g). Part I was fractioned on a silica gel column (200–300 mesh, Qingdao Haiyang Chem. Co., Ltd.) using gradient PE-EtOAc solvent system to afford forty fractions (XMZ-1 to XMZ-40). XMZ-10 (0.3 g) was isolated by silica gel column (400 mesh, Qingdao Haiyang Chem. Co., Ltd.) with gradient PE-EtOAc solvent system, then followed by HPLC on an Agilent C-8 column (Agilent Zorbax Eclipse XDB C-8, 250 × 9.4 mm, 5 ␮m, MeOH:H2 O 80:20, 4 ml/min) to provide 7 (44.4 mg). XMZ-12 (4.2 g) was purified by repeated silica gel column (400 mesh) with gradient light petroleum and EtOAc to give 3 (5.9 mg). XMZ-30 (0.7 g) was similarly purified by repeated silica gel column and followed by HPLC on Agilent C-8 column (MeOH:H2 O 80:20, 4 ml/min) to give 8 (37.9 mg). Combined fractions (1.6 g) of XMZ-22 to XMZ-29 was introduced to a silica gel column (400 mesh) and eluted by gradient PE-EtOAc system (3:1 to pure EtOAc) to give 9 (23.2 mg). 10 (95.7 mg), 11 (40 mg) and 12 (8.9 mg) were isolated and purified from XMZ-33 (2.5 g) by repeated silica gel column (400 mesh) with gradient CHCl3 -CH3 OH system. XMZ-35 (3.1 g) was separated by HPLC on YMC-Pack ODSA column (250 × 20 mm, 5 ␮m, MeOH:H2 O 35:65, 10 ml/min) to obtain 1 (4.4 mg). XMZ36 was re-chromatographed on a silica gel column (gradient PE-EtOAc 10:1 to 2:8), followed by HPLC on YMC-Pack ODS-A column (MeOH:H2 O 2:8, 10 ml/min) to obtain 6 (12 mg). Part II was chromatographed on a silica gel column (200–300 mesh) using PE-EtOAc gradient (50:1 to 1:5) to afford 31 fractions (SWJ-1 to SWJ-31). SWJ-12 (4.2 g) was subjected to a silica gel column (400 mesh) with gradient PE-EtOAc solvent system (5:1 to 1:4) to yield 2 (9.6 mg), 4 (9.2 mg) and 5 (8.7 mg).

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Cytotoxicity assay

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The cytotoxic activities of all compounds were evaluated by MTT method to against three human cancer cell lines: Hela, MCF-7 and A549, using Doxorubicin as positive control. Cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum in 5% CO2 at 37 ◦ C. Cells (100 ␮l) were distributed evenly into 96well microplates, containing 5 × 103 cells per well, and incubated

for 12 h. Designated wells were treated with DMSO solutions of tested compound at various concentrations (0.0625, 0.32, 1.6, 8 and 40 ␮g/ml) in triplicate. After 48 h incubation, MTT was added to each well to incubate an additional 4 h. After treatment, the absorbance of each well was read at 490 nm by an enzyme-labeled detector (BioTek ELx800). IC50 values to different cell lines were determined by the dose–response curve (Yu et al., 2016). Statistical analysis Statistical significance was determined with the standard deviation generally less than 10%, and all values were carried out using the t-test (SPSS 12.0). Results and discussion In present phytochemical investigation on searching for bioactive agents from this plant, the extracting method was optimized to afford two parts, part I was from acidic solution of EtOH extracts, part II was from basic solution. Chromatographic separation of these two parts, twelve compounds were obtained, containing nine isolates from part I, and three isolates from part II. The chemical structures were identified mainly by spectroscopic analysis to be (−)-8-oxodiscretamine (1) (Shono et al., 2016), N-formylannonaine (2) (Yusoff et al., 2014), isooncine (3) (Wu et al., 1990), isoursuline (4) (Chen et al., 2000), cleistopholine (5) (Wang et al., 2011), northalifoline (6) (Lee et al., 2010), N-2-phenylethylcinnamamide (7) (Gu et al., 2013), N-transcinnamoyltyramine (8) (Wang et al., 2012a,b), binaphthalene-2phenol-3-aldehyde (9) (Wu et al., 1989), arborone (10) (Tulake et al., 2012), syringaresinol (11) (Mo and Mai, 2012), balanophonin (12) (Wang et al., 2010), including eight alkaloids (1–8), three lignans (10–12) and one gossypol derivative (9). It should be noted that all isolates were reported from this plant for the first time. Among them, binaphthalene-2-phenol-3aldehyde (9) was firstly isolated from the natural resources, and its complete 1 H NMR and 13 C NMR were assigned by 2D NMR. N-formylannonaine (2) exists as a mixture of two conformational isomers (Z/E = 1/2.5, calculated by the integration of 1 H NMR) in deuterated methanol. A single crystal of isoursuline (4) was firstly recrystalized, and the crystal structure had been determined by single-crystal X-ray diffraction method (Niu et al., 2015). The X-ray data were deposited in the Cambridge Crystallographic Data Centre with number of CCDC-1494107.

Fig. 1. The inhibition ratio (IR, %) of six cytotoxical compounds against three cancer cell lines at various concentrations. IR values were calculated using the formula: IR (%) = [OD (control group) − OD (administration group)]/OD (control group) × 100%. *p < 0.05, **p < 0.01.

Please cite this article in press as: Wang, T., et al. Chemical constituents from ethanol extract of Polyalthia rumphii branches and their cytotoxicity evaluation. Revista Brasileira de Farmacognosia (2017), https://doi.org/10.1016/j.bjp.2018.03.004

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Partial tested compounds showed cytotoxicities inhibiting three human cancer cell lines in vitro (Fig. 1): A considerable increase of inhibition ratio (IR, %) data was demonstrated with the increase in the concentrations. IR values more than 50% were only at high dose of 40 ␮g/ml, those who achieved more than 50% of IR were 4 to Hela of 50.6% (p < 0.01), 6 to Hela of 53.7% (p < 0.01), 7 to Hela of 51.5% (p < 0.01), 8 to Hela of 54.7% (p < 0.01), 12 to Hela of 51.1% (p < 0.01), 6 to MCF-7 of 53.8% (p < 0.01), 6 to A549 of 50.3% (p < 0.01). While all of compounds at low dose of 0.0625 ␮g/ml were failed to inhibit the proliferation of the tested cell lines. In order to evaluate the cytotoxicities, the IR data were used to calculate the IC50 values by the dose–response curves, which were presented in Table 1. Among the tested isolates, five alkaloids (2, 4, 6, 7 and 8) and one lignan (12) demonstrated full or partial activities against these tested cancer cell lines ranging from 25 to 40 ␮g/ml. Among these active alkaloids, 6 and 8 demonstrated broad-spectrum active to all these tested cell lines, both of which were with amide functional group. Furthermore, all active compounds showed antiproliferative action to Hela cell lines. (−)-8-Oxodiscretamine (1) Brown, amorphous powder (4.4 mg). [␣]D : −37.3 (c 0.15, MeOH). ESI-MS m/z: 364.2 [M+Na]+ . HRMS-ESITOF m/z: [M+H]+ calcd for C19 H20 NO5 : 342.1336, found: 342.1350. 1 H NMR (CDCl , 400 MHz) ı: 6.67 (H-1, s), 6.77 (H-4, s), 2.73 (H3 5␣, m), 2.91 (H-5␤, m), 2.91 (H-6␣, m), 4.96 (H-6␤, d, J 10.4 Hz), 7.08 (H-11, d, J 8.0 Hz), 6.92 (H-12, d, J 8.0 Hz), 3.02 (H-13␣, m), 2.86 (H-13␤, m), 4.77 (H-14, d, J 12.8 Hz), 3.91 (2-OCH3 , s), 4.00 (9-OCH3 , s). 13 C NMR (CDCl3 , 100 MHz) ı: 108.3 (C-1), 145.7 (C-2), 144.5 (C-3), 114.4 (C-4), 128.2 (C-4a), 29.22 (C-5), 38.4 (C-6), 162.3 (C-8), 121.6 (C-8a), 147.4 (C-9), 149.0 (C-10), 118.2 (C-11), 122.8 (C-12), 130.7 (C-12a), 39.2 (C-13a), 55.2 (C-14), 127.1 (C-14a), 56.2 (2-OCH3 ), 62.4 (9-OCH3 ). N-formylannonaine (2) Yellow solid (9.6 mg). ESI-MS m/z (+): 294.1 [M+H]+ , 316.1 [M+Na]+ . IR (KBr) cm−1 : 2926, 2860, 1645, 1585, 1470, 1408, 1260, 1203, 1116, 1044, 972. Z-isomer: 1 H NMR (CDCl3 , 400 MHz) ı: 6.59 (H-3), 2.90 (H-4ax ), 2.69 (H-4eq ),

Table 1 Q5 The active isolates and IC50 values of cytotoxic activities. Isolates

2 4 6 7 8 12 Doxorubicin a

Inactive at IC50 > 40 ␮g/ml.

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A549

32.3 35.5 32.3 29.7 26.9 37.0 0.5

>40a 36.9 32.9 36.7 36.2 >40 0.9

>40 38.0 39.3 >40 33.4 >40 1.4

3.41 (H-5ax ), 3.85 (H-5eq ), 5.05 (H-6a), 2.84 (H-7ax ), 3.24 (H-7eq ), 7.23–7.34 (H-8,9,10), 8.10 (H-11), 5.99/6.11 (OCH2 O), 8.27 (CHO). 13 C NMR (CD OD, 100 MHz) ı: 143.3 (C-1), 147.1 (C-2), 107.5 (C-3), 3 126.6 (C-3a), 31.0 (C-4), 42.2 (C-5), 49.5 (C-6a), 33.6 (C-7), 135.2 (C-7a), 128.1 (C-8), 127.6 (C-9), 126.6 (C-10), 128.9 (C-11), 130.5 (C-11a), 117.5 (C-11b), 124.7 (C-11c), 101.0 (OCH2 O), 162.1 (CHO); E-isomer: 1 H NMR (CDCl3 , 400 MHz) ı: 6.62 (H-3), 2.75 (H-4ax ), 2.75 (H-4eq ), 3.10 (H-5ax ), 4.47 (H-5eq ), 4.63 (H-6a), 3.16 (H-7ax ), 2.90 (H-7eq ), 7.23–7.34 (H-8,9,10), 8.11 (H-11), 5.99/6.11 (OCH2 O), 8.40 (CHO). 13 C NMR (CD3 OD, 100 MHz) ı: 143.1 (C-1), 147.6 (C-2), 107.9 (C-3), 126.6 (C-3a), 29.7 (C-4), 36.2 (C-5), 53.3 (C-6a), 37.8 (C-7), 134.5 (C-7a), 128.9 (C-8), 128.0 (C-9), 127.1 (C-10), 128.5 (C11), 130.3 (C-11a), 117.0 (C-11b), 124.1 (C-11c), 101.1 (OCH2 O), 162.1 (CHO). Isooncine (3) Yellow amorphous solid (5.9 mg). ESI-MS m/z (+): 241.1 [M+H]+ . IR (KBr) cm−1 : 3435, 2925, 2825, 1740, 1640, 1463, 1383, 1252, 1119. 1 H NMR (CDCl3 , 400 MHz) ı: 8.31 (H-3, d, J 5.2 Hz), 7.36 (H-5, s), 7.22 (H-8, s), 6.87 (H-2, d, J 5.2 Hz), 3.99 (6OMe, s), 2.58 (1-Me, s). Isoursuline (4) Yellow single crystal (9.2 mg). ESI-MS m/z (+): 242.2 [M+H]+ , 264.2 [M+Na]+ . IR (KBr) cm−1 : 3434.6, 2925.6, 1625.3, 1570.6, 1466.5. 1 H NMR (CD3 OD, 400 MHz) ı: 2.95 (1-Me, s), 7.05 (H-2, J 5.6 Hz), 8.32 (H-3, J 5.6 Hz), 3.97 (6-OMe, s), 6.94 (H7, J 8 Hz), 7.22 (H-8, J 8 Hz). 13 C NMR (CD3 OD, 100 MHz) ı: 149.4 (C-1), 17.3 (1-Me), 126.3 (C-2), 152.0 (C-3), 165.8 (C-4a), 126.5 (C4b), 144.7 (C-5), 156.0 (C-6), 57.2 (6-OMe), 113.6 (C-7), 118.2 (C-8), 129.2 (C-8a), 192.8 (C-9), 127.7 (C-9a). Cleistopholine (5) Yellow amorphous solid (8.7 mg). ESI-MS m/z (+): 224.1 [M+H]+ , 246.1 [M+Na]+ , 469.2 [2M+Na]+ . IR (KBr) cm−1 : 2930, 1682, 1591, 1460, 1414, 1334, 1301, 1125. 1 H NMR (CD3 OD, 400 MHz) ı: 8.90 (H-2, d, J 4.8 Hz), 7.50 (H-3, dd, J 4.8, 0.8 Hz), 8.37 (H-5, m), 7.83 (H-6, m), 7.83 (H-7, m), 8.27 (H-8, m), 2.92 (22.8-Me, s). 13 C NMR (CD3 OD, 100 MHz) ı: 153.3 (C-2), 131.4 (C-3), 151.5 (C4), 129.2 (C-4a), 127.5 (C-5), 134.6 (C-6), 134.2 (C-7), 127.3 (C-8), 129.0 (C-8a), 185.0 (C-9), 150.1 (C-9a), 181.9 (C-10), 132.0 (C-10a). Northalifoline (6) Amorphous solid (12 mg). ESI-MS: m/z 216.0 [M+Na]+ , 194.0 [M+H]+ . 1 HNMR (400 MHz, CD3 OD) ıH (ppm): 7.43 (1H, s, H-8), 6.65 (1H, s, H-5), 3.84 (3H, s, 4-OCH3 ), 3.41 (2H, t, J 6.8, H-3), 2.80 (2H, t, J 6.8, H-4). 13 CNMR (100 MHz, CD3 OD) ıC (ppm): 168.8 (C-1), 152.1 (C-6), 148.2 (C-7), 135.3 (C-4a), 121.1 (C-8a), 115.0 (C-8), 111.5 (C-5), 56.5 (6-OCH3 ), 41.1 (C-3), 28.5 (C-4). N-2-phenylethylcinnamamide (7) Amorphous solid (44.4 mg). ESI-MS: m/z 252.4 [M+H]+ , 274.3 [M+Na]+ . 1 H NMR (400 MHz, CDCl3 ) ıH (ppm): 7.62 (1H, d, J 15.6 Hz, H-7 ), 7.49(2H, dd, J 8.0, 3.2 Hz, H-2 , 6 ), 7.36 (3H, m, H-3 ,4 ,5 ), 7.24 (5H, m, H-2,3,4,5,6), 6.32 (1H, d, J 15.6 Hz, H-8 ), 3.66 (2H, q, J 6.8 Hz, H-8), 2.90 (2H, t, J 6.8 Hz, H-7), 5.64 (1H, brs, NH ). 13 C NMR (CDCl3 , 100 MHz) ıC (ppm): 165.8 (C-9), 141.1 (C-7 ), 138.9 (C-1 ), 134.8 (C-1), 129.9

Please cite this article in press as: Wang, T., et al. Chemical constituents from ethanol extract of Polyalthia rumphii branches and their cytotoxicity evaluation. Revista Brasileira de Farmacognosia (2017), https://doi.org/10.1016/j.bjp.2018.03.004

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(C-4 ), 128.79 (C-3 ,5 ), 128.81 (C-2,6), 128.7 (C-2 ,6 ), 127.8 (C-3,5), 126.6 (C-4), 120.5 (C-8 ), 40.8 (C-8), 35.6 (C-7). N-trans-cinnamoyltyramine (8) Amorphous solid (37.9 mg). ESIMS: m/z 268.3 [M+H]+ , 290.2 [M+Na]+ . 1 H NMR (400 MHz, CD3 OD) ıH (ppm): 7.54 (1H, d, J 15.8 Hz, H-7 ), 7.51 (2H, m, H-2 , 6 ), 7.37 (3H, m, H-3 , 4 , 5 ), 7.06 (2H, d, J 8.3 Hz, H-2, 6), 6.72 (2H, d, J 8.3 Hz, H-3, 5), 6.56 (1H, d, J 16.0 Hz, H-8 ), 3.48 (2H, t, J 7.8 Hz, H-8), 2.77 (2H, t, J 7.6 Hz, H-7), 4.60 (1H, brs, NH ). 13 C NMR (100 MHz, CD3 OD) ıC (ppm): 131.3 (C-1), 130.8 (C-2, 6), 116.3 (C-3, 5), 157.0 (C-4), 35.8 (C-7), 42.6 (C-8), 136.4 (C-1 ), 128.9 (C-2 , 6 ), 130.7 (C-3 , 5 ), 130.8 (C-4 ),141.7 (C-7 ), 122.0 (C-8 ), 168.7 (C-9 ). Binaphthalene-2-phenol-3-aldehyde (9) Yellow amorphous solid (23.2 mg). ESI-MS: m/z 365.0 [M+Na]+ , HRMS-ESI-TOF (m/z 365.0806 [M+Na]+ , calcd. for 365.0784). 1 H NMR (400 MHz, CDCl3 ) ıH (ppm): 8.38 (2H, s, H-4,4 ), 8.05 (2H, d, J 7.2,2.0 Hz, H-5,5 ), 7.45 (4H, m, H-6,6 ,7,7 ), 7.24 (2H, dd, J 7.2,2.0 Hz, H-8,8 ), 10.23 (2H, s, H-9,9 ), 10.61 (2H, s, -OH); 13 C NMR (100 MHz, CDCl3 ) ıC (ppm): 115.6 (C-1,1 ), 152.7 (C-2,2 ), 121.1 (C-3,3 ), 137.4 (C-4,4 ), 137.4 (C4a,4a ), 129.0 (C-5,5 ), 129.6 (C-6,6 ), 123.9 (C-7,7 ), 123.5 (C-8,8 ), 126.7 (C-8a, 8a ), 195.7 (C-9,9 ). Arborone (10) Amorphous solid (95.7 mg). ESI-MS: m/z 485.2 [M+Na]+ , 947.3 [2M+Na]+ . 1 H NMR (400 MHz, CDCl3 ) ıH (ppm): 7.46 (2H, s, H-2,6), 6.55 (2H, s, H-2 ,6 ), 5.08 (1H, d, J 5.6 Hz, H-7 ), 4.42 (1H, m, H␤ -9), 4.35 (1H, m, H-8), 4.28 (1H, dd, J 7.6, 2.4 Hz, H␣ -9), 3.93 (3H, s, 4-OCH3 ), 3.92 (6H, s, 3,5-OCH3 ), 3.84 (6H, s, 3 ,5 -OCH3 ), 3.83 (3H, s, 4 -OCH3 ), 3.42 (2H, d, J 6.8 Hz, H-9 ), 2.98 (1H, m, H-8 ); 13 C NMR (100 MHz, CDCl ) ı (ppm): 198.5 (C-7), 153.2 (C-3,5), 3 C 153.0 (C-3 ,5 ), 142.8 (C-4), 136.9 (C-4 ), 133.6 (C-1 ), 131.1 (C-1), 106.4 (C-2,6), 102.5 (C-2 ,6 ), 81.3 (C-7 ), 68.7 (C-9), 61.7 (C-9 ), 60.8 (4-OCH3 ), 60.7 (4 -OCH3 ), 56.2 (3,5-OCH3 ), 56.0 (3 ,5 -OCH3 ), 49.7 (C-8 ), 48.6 (C-8). Syringaresinol (11) Amorphous solid (40 mg). ESI-MS: m/z 441.1 [M+Na]+ , 417.1 [M−H]− . 1 H NMR (400 MHz, CDCl3 ) ıH (ppm): 6.56 (4H, s, H-2,2 ,6,6 ), 4.74 (2H, d, J 3.5 Hz, H-7,7 ), 3.35 (2H, m, H8,8 ), 3.58 (4H, d, J 8 Hz, H-9,9 ), 3.88 (12H, s, 3,3 ,5,5 -OCH3 ). 13 C NMR (100 MHz, CDCl3 ) ıC (ppm): 131.1 (C-1,1 ), 101.0 (C-2,2 ,6,6 ), 146.2 (C-3,3 ,5,5 ), 133.4 (C-4,4 ), 146.2 (C-3,3 ,5,5 ), 85.1 (C-7,7 ), 53.3 (C-8,8 ), 70.8 (C-9,9 ), 55.4 (3,3 ,5,5 -OCH3 ). Balanophonin (12) Amorphous solid (8.9 mg). ESI-MS: m/z 379.1 [M+Na]+ , 355.1 [M−H]− . 1 H NMR (400 MHz, CDCl3 ) ıH (ppm): 9.64 (1H, d, J 7.6 Hz, H-9 ), 7.41 (1H, d, J 15.6 Hz, H-7 ), 7.13 (1H, brs, H-6 ), 7.04 (1H, brs, H-2 ), 6.89 (1H, brs, H-2), 6.88 (2H, m, H-5,6), 6.60 (1H, dd, J 15.6,7.6 Hz, H-8 ), 5.64 (1H, d, J 6.8 Hz, H-7), 3.97 (2H, m, H-9), 3.93 (3H, s, 3 -OCH3 ), 3.87 (1H, s, 3-OCH3 ), 3.68 (1H, q, J 6.0 Hz, H-8). 13 C NMR (100 MHz, CDCl3 ) ıC (ppm): 193.7 (C-9 ), 153.1 (C-7 ), 151.1 (C-4 ), 146.7 (C-3), 145.9 (C-4), 144.8 (C-3 ), 132.2 (C-5 ), 129.1 (C-1), 128.1 (C-1 ), 126.4 (C-8 ), 119.4 (C-6), 118.2 (C6 ), 114.5 (C-5), 112.2 (C-2 ), 108.7 (C-2), 89.0 (C-7), 63.9 (C-9), 56.1 (3 -OCH3 ), 56.0 (3-OCH3 ), 53.0 (C-8).

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TSW and XPW contributed equally to this paper on structural determination, writing this paper and running the laboratory work;

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Please cite this article in press as: Wang, T., et al. Chemical constituents from ethanol extract of Polyalthia rumphii branches and their cytotoxicity evaluation. Revista Brasileira de Farmacognosia (2017), https://doi.org/10.1016/j.bjp.2018.03.004

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