Characterization of Antiproliferative Activity Constituents from

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Characterization of Antiproliferative Activity Constituents from Artocarpus heterophyllus Zong-Ping Zheng, Yang Xu, Chuan Qin, Shuang Zhang, Xiaohong Gu, Yingying Lin, Guobin Xie, Mingfu Wang, and Jie Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf500159z • Publication Date (Web): 22 May 2014 Downloaded from http://pubs.acs.org on June 3, 2014

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Journal of Agricultural and Food Chemistry

Characterization of Antiproliferative Activity Constituents from Artocarpus heterophyllus Zong-Ping Zheng†,ǁ, Yang Xu ‡,ǁ, Chuan Qin†, Shuang Zhang†, Xiaohong Gu†, Yingying Lin‡, Guobin Xie‡, Mingfu Wang§, Jie Chen*,†,⊥ †

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi,

Jiangsu, P.R. China ‡

School of Pharmaceutical Sciences, Xiamen University, Xiamen, Fujian, P.R. China

§

School of Biological Sciences, The University of Hong Kong, Hong Kong, P.R. China

⊥

Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi,

Jiangsu, P.R. China

ACS Paragon Plus Environment


1

ABSTRACT

2

Artocarpus heterophyllus is an evergreen fruit tree cultivated in many tropical regions.

3

Our previous studies have shown that some of its compositions exhibited potential

4

tyrosinase inhibition activities. In this study, we indentified eight new phenolic

5

compounds, artoheterophyllin E-J (1-6), 4-geranyl-2',3,4',5-tetrahydroxy-cis-stilbene (7),

6

5-methoxy-morican M (8), two new natural compounds (9-10), 2,3-dihydro-5,7-

7

dihydroxy-2-(2-hydroxy-4-methoxyphenyl)-4H--benzopyran-4-one and 6-[(1S,2S)-1,2-

8

dihydroxy-3-methylbutyl]-2-(2,4-dihydroxyphenyl)-5-hydroxy-7-methoxy-3-(3-methyl-

9

2-buten-1-yl)-4H-1-benzopyran-4-one, together with twenty-three known compounds

10

(11-33), from the ethanol extract of the wood of A. heterophyllus. The structures of eight

11

new compounds (1-8) and two new natural compounds were established by extensive 1D-

12

and 2D-NMR experiments. The anticancer effects of the isolated compounds were

13

examined in MCF-7, H460 and SMMC-7721 human cancer cell lines by MTT assay.

14

Compounds 5, 11, 12, and 30 significantly reduced the cell viabilities of these cell lines.

15

Especially, compound 11 and 30 resulted in more potent cytotoxicity than the positive

16

control, 5-fluorouracil (5-Fu) in SMMC-7721 cell line, with IC50 values 15.85 and 12.06

17

µM, while compound 30 exhibited more potent cytotoxicity than 5-Fu in NCI-H460 cell

18

line, with IC50 value 5.19 µM. In addition, this study suggested that compounds 11 and 30

19

from the wood of A. heterophyllus have anticancer potential via MAPK pathways.

20

KEYWORDS: Artocarpus heterophyllus; phenolic compounds; anticancer activity

21 22 23


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INTRODUCTION

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Mitogen-activated protein kinases (MAPKs) are serine-threonine kinases which include

26

extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK).

27

MAPKs pathways regulate intracellular signaling including cell apoptosis, proliferation,

28

differentiation, survival and death.1 It is a very complicated function that ERKs, JNKs

29

and p38 MAPKs play a role in cancer development. These MAPKs pathways could

30

antagonize cell proliferation, survival and invasion in some type of cells, whereas they

31

could also promote the morphological transformation and cell proliferation in cancer cells.

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The functions of MAPKs are accordingly dependent on the different cells’ responses.2-5

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Especially, these MAPK pathways are expected to provide new strategies for anticancer

34

therapeutic approaches.1 Therefore, screening selective regulators to different types of

35

cancer cell lines is likely to be a better way to cancer therapies targeted against MAPK

36

pathways.

37

Artocarpus heterophyllus (Jackfruit) is an evergreen fruit tree belonging to the

38

Artocarpus genus of the family Moraceae, found in tropical and subtropical regions. A.

39

heterophyllus is widely cultivated in tropical regions of India, Bangladesh, Nepal, Sri

40

Lanka, Vietnam, Thailand, Malaysia, Indonesia, Philippines, Africa, and Brazil.6 It is also

41

an important tropical fruit in southern China, distributed and cultivated in Hainan,

42

Guangdong, Guangxi, and Yunnan Provinces in China.7 Previous studies suggested that A.

43

heterophyllus was rich in phenolic compounds, flavonoids, stilbenoids, arylbenzofurans

44

and possessed widely medicinal uses including antioxidant, anti-inflammatory,

45

antibacterial, antifungal, antineoplastic and hypoglycemic effects.8-13 Furthermore, the

46

extracts and chemicals prepared from A. heterophyllus were also found to exhibit



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cytotoxic effects on T47D, SMMC-7721, SGC-7901, hepatoma, choriocarcinoma, and

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melanoma cell lines in vitro.14-18 However, little information is available on the

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systematic study of the phenolic compounds from A. heterophyllus on cytotoxic effects of

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MCF-7, SMMC-7721 and NCI-H460 Cells and the pathways. Therefore, we used

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chromatography to isolate the active components and used NMR and MS to identify the

52

compounds, combined with cytotoxic activity assay to confirm the in vitro anti-cancer

53

effects of A. heterophyllus.

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MATERIALS AND METHODS

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General Experimental Procedures. Sephadex LH-20 was purchased from GE

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Healthcare Bio-Sciences AB (Uppsala, Sweden). Silica gel (200-300 mesh) for column

58

chromatography and TLC plates ( HSGF254) were purchased from Yantai Jiangyou

59

Silicone Development Co., LTD. (Yantai, PR China). Amberlite XAD16 was purchased

60

from Sigma Chemical Co (St. Louis, USA). Semi-preparative HPLC system was carried

61

on a Waters 600 system equipped with a 2487 dual-wavelength detector, a Masslynx

62

V4.0 software and a YMC C18 (2) column (250 × 20 mm, 5 µm). 1H NMR,

63

DEPT, HMQC, and HMBC spectra were obtained on a Bruker 400 NMR spectrometer.

64

Molecular weights of compounds were analyzed on Waters Maldi Syapt Q-Tof mass

65

spectrometer. Cell morphologies were observed by ZEISS LSM 5 EXCITER and ZEISS

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AXIO IMAGER A1 microscopes. Antibodies, inhibitors and other chemicals, except

67

otherwise noted, were purchased from Sigma-Aldrich Chemicals.

13

C NMR,

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Plant Material. The wood of A. heterophyllus was collected at Wenchang county,

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Hainan Province, PR China, in May 2012. The voucher specimens (accession number


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70

20120501) were deposited at State Key Laboratory of Food Science and Technology,

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Jiangnan University.

72

Extraction and Isolation. The wood of Artocarpus heterophyllus (10 kg) was batched

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twenty times with 95% ethanol (3 × 2 L) under ultrasound condition. The extract was

74

concentrated under vacuum at 45 oC with a rotor evaporator, and 272.2 g crude extract

75

was offered. The crude extract was separated with an Amberlite XAD16 column (eluted

76

with 20, 40, 60, 80, and 95% ethanol) to offer six frictions (Fr.1-Fr.6). Fr.1 (5.5 g, 20%

77

ethanol fraction) was subjected to sephadex LH-20 and was eluted by MeOH-H2O (1:1)

78

to offer two fractions (Fr.1A-1B). Fr.1A (4.2 g) was purified by silica gel and was eluted

79

by ethyl acetate-hexane (1:1) to offer 19 (3.8 g). Fr.1B (86 mg) was further separated by

80

preparative HPLC (eluted by 45% MeOH) to offer 27 (28 mg). Fr.2 (2.0 g, 40% ethanol

81

fraction) was subjected by silica gel and was eluted by ethyl acetate-hexane (3:1), then it

82

was separated by sephadex LH-20 [eluted by MeOH-H2O (1:1)] to offer 1 (8.8 mg) and

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another fraction (Subfr.A). Subfr.A was further purified by HPLC to obtain 32 (1.9 mg)

84

and 33 (3.4 mg). Fr.3 (12.7 g, 60% ethanol fraction) was separated by silica gel [CH2Cl2-

85

MeOH (20:1)] to give three fractions (Fr.3A-3C). Fr.3A was further purified by sephadex

86

LH-20 [MeOH-H2O (1:1)] to get 14 (752 mg). Fr.3B was separated by sephadex LH-20

87

[MeOH-H2O (1:1)] to offer 16 (2596.4 mg), 22 (6.5 mg), 26 (8.4 mg), 21 (399.3 mg) and

88

8 (3.1 mg). Fr.3C was separated by sephadex LH-20 [MeOH-H2O (1:1)] to offer two

89

fractions (Subfr.B-C), Subfr.B was purified by silica gel [CH2Cl2-MeOH (25:1)] to give

90

28 (14.6 mg). Subfr.C was separated by silica gel [CH2Cl2-MeOH (30:1)] and HPLC

91

(47% MeOH) to offer 3 (1.5 mg). Fr.4 (15.8 g, 60% ethanol fraction) was separated by

92

silica gel [hexane-ethyl acetate (3:1)] to give five fractions (Fr.4A-4E). Fr.4A was



93

purified by sephadex LH-20 [MeOH-H2O (1:1)] to offer 20 (2059.3 mg). Fr.4B was

94

separated by sephadex LH-20 [MeOH-H2O (1:1)] to get 23 (24.6 mg). Fr.4C was purified

95

by sephadex LH-20 [MeOH-H2O (1:1)] to obtain 24 (185 mg), 29 (18 mg), and another

96

fraction (Subfr.D). Subfr.D was purified by HPLC (60% MeOH) to obtain 7 (13.7 mg).

97

Fr.4E was separated by sephadex LH-20 [MeOH-H2O (1:1)] to offer 9 (88.8 mg). Fr.5

98

(24.3 g, 80% ethanol fraction) was separated by silica gel and eluted by CH2Cl2, CH2Cl2-

99

MeOH (50:1), and CH2Cl2-MeOH (25:1) to offer three fractions (Fr.5A-5C). Fr.5A was

100

further purified by silica gel (eluted by CH2Cl2) to get 25 (17.4 mg) and 31 (2.6 mg).

101

Fr.5B was separated by silica gel [eluted by CH2Cl2-MeOH (50:1)] to obtain 6 (4.3 mg).

102

Fr.5C was separated by sephadex LH-20 [MeOH-H2O (3:2)] to offer three fractions

103

(Subfr.E-G). Subfr.E was purified by silica gel [CH2Cl2-MeOH (50:1)] and then further

104

separated by HPLC (72% MeOH) to give 10 (0.4 mg), 4 (0.9 mg), and 5 (1.6 mg).

105

Subfr.F was separated by silica gel [CH2Cl2-MeOH (50:1)] to get 30 (210.8 mg) and

106

another fraction (Subfr.H). Subfr.H was separated by sephadex LH-20 [MeOH-H2O (1:1)]

107

to get 15 (56.8 mg) and 18 (48.6 mg). Subfr.G was purified by silica gel [CH2Cl2-MeOH

108

(25:1)] to obtain 2 (6.4 mg). Fr.6 (62 g, 95% ethanol fraction) was separated by silica gel

109

and was eluted by CH2Cl2 to give 11 (28.9 g), 12 (16.9 g), 13 (1.2 g), 17 (69 mg).

110

Artoheterophyllin E (1). Brown amorphous powder: C15H12O5; [α]20D +0.005 (c 0.002,

111

acetone); UV (MeOH) 236, 277 nm; IR (KBr) νmax 3379, 1612, 1463, 1111, 1007 cm-1;

112

1

113

OH-4'), 7.08 (1H, d, J = 8.4 Hz, H-6), 6.31 (1H, dd, J = 8.4, 2.4 Hz, H-5), 6.18 (1H, d, J

114

= 2.4 Hz, H-3), 5.94 (1H, d, J = 2.0 Hz, H-5'), 5.69 (1H, d, J = 2.0 Hz, H-3'), 5.20 (1H, m,

115

H-1''), 5.15 (1H, m, H-3''), 2.12 (2H, m, H-2''); 13C NMR (Acetone-d6, 100 MHz) δ: 160.3

H NMR (Acetone-d6, 400 MHz) δ: 9.28 (1H, s, OH-6'), 9.12 (1H, s, OH-4), 8.91 (1H, s,


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(C, C-4'), 160.2 (C, C-4), 158.2 (C, C-6'), 155.8 (C, C-2, 2'), 132.4 (CH, C-6), 114.0 (C,

117

C-1), 108.9 (CH, C-5), 103.6 (CH, C-3), 102.4 (C, C-1'), 96.0 (CH, C-5'), 95.0 (CH, C-3'),

118

67.8 (CH, C-1''), 62.8 (CH, C-3''), 27.8 (CH2, C-2''). HR-ESI-MS m/z 273.0760 [M+H]+

119

(calcd. for C15H13O5 273.058).

120

Artoheterophyllin F (2). Brown powder: C24H26O4; [α]20D -0.008 (c 0.0001, CHCl3);

121

UV (MeOH) 246, 324 nm; IR (KBr) νmax 3376, 2964, 1605, 1510, 1447, 1159 cm-1; 1H

122

NMR (Acetone-d6, 400 MHz) δ: 8.57 (1H, OH-2'), 8.42 (1H, s, OH-5), 8.39 (1H, s, OH-

123

4'), 7.40 (1H, d, J = 8.4 Hz, H-6'), 7.35 (1H, d, J = 16.4 Hz, H-7'), 6.87 (1H, d, J = 16.4

124

Hz, H-8'), 6.71 (1H, d, J = 10.0 Hz, H-1''), 6.57 (1H, d, J = 1.2 Hz, H-2), 6.49 (1H, s, H-

125

6), 6.44 (1H, d, J = 2.4 Hz, H-3'), 6.39 (1H, dd, J = 8.4, 2.4 Hz, H-5'), 5.58 (1H, d, J =

126

10.0 Hz, H-2''), 5.13 (1H, m, H-6''), 2.14 (2H, m, H-4''), 1.71 (2H, m, H-5''), 1.64 (3H, s,

127

H-9''), 1.57 (3H, s, H-8''), 1.37 (3H, s, H-10''); 13C NMR (Acetone-d6, 100 MHz) δ: 159.3

128

(C, C-4'), 157.1 (C, C-2'), 155.3 (C, C-3), 154.1 (C, C-5), 140.7 (C, C-1), 132.0 (C, C-7''),

129

128.4 (CH, C-6'), 127.8 (CH, C-2''), 126.1 (CH, C-8'), 125.2 (CH, C-6''), 124.5 (CH, C-

130

7'), 118.4 (CH, C-1''), 117.3 (C, C-1'), 109.4 (C, C-4), 108.6 (CH, C-5'), 106.4 (CH, C-2),

131

106.3 (CH, C-6), 103.7 (CH, C-3'), 78.7 (C, C-3''), 41.9 (CH2, C-4''), 26.6 (CH3, C-10''),

132

25.9 (CH3, C-9''), 23.5 (CH2, C-5''), 17.7 (CH3, C-8''). HR-ESI-MS: m/z 379.1905

133

[M+H]+ (calcd. for C24H27O4 379.1904).

134

Artoheterophyllin G (3). Yellow powder: C20H18O7; UV (MeOH) 253 nm; 1H NMR

135

(CD3OD, 400 MHz) δ: 7.08 (1H, d, J = 8.8 Hz, H-6'), 6.41 (1H, s, H-3'), 6.40 (1H, dd, J =

136

8.8, 2.3 Hz, H-5'), 6.26 (1H, d, J = 2.0 Hz, H-8), 6.18 (1H, d, J = 2.0 Hz, H-6), 5.39 (1H,

137

br t, J = 6.0 Hz, H-2''), 3.82 (2H, s, H-4''), 3.14 (2H, d, J = 7.2 Hz, H-1''), 1.46 (3H, s, H-

138

5''); 13C NMR (CD3OD, 100 MHz) δ: 184.3 (C=O, C-4), 166.3 (C, C-7), 164.4 (C, C-2),



139

164.0 (C, C-5), 162.7 (C, C-9), 160.4 (C, C-4'), 158.4 (C, C-2'), 137.0 (C, C-3''), 133.1

140

(CH, C-6'), 124.8 (CH, C-2''), 122.2 (C, C-3), 114.0 (C, C-1'), 108.8 (CH, C-5'), 104.6

141

(CH, C-3'), 106.1 (C, C-10), 100.3 (CH, C-6), 95.3 (CH, C-8), 69.6 (CH2, C-4''), 25.3

142

(CH2, C-1''), 14.3 (CH3, C-5''). HR-ESI-MS: m/z 371.1126 [M+H]+ (calcd. for C20H19O7

143

371.1125).

144

Artoheterophyllin H (4). Yellow powder: C26H26O8; UV (MeOH) 277 nm; 1H NMR

145

(CD3OD, 400 MHz) δ: 7.08 (1H, d, J = 8.0 Hz, H-6'), 6.76 (1H, d, J = 16.4 Hz, H-1''),

146

6.68 (1H, d, J = 16.4 Hz, H-2''), 6.56 (1H, s, H-8), 6.41 (1H, overlapped, H-3'), 6.40 (1H,

147

overlapped, H-5'), 5.10 (1H, t, J = 6.0 Hz, H-2'''), 3.93 (3H, s, OCH3), 3.90 (3H, s, OCH3),

148

3.11 (2H, d, J = 7.2 Hz, H-1'''), 1.59, 1.40 (each 3H, s, H-4''', 5'''), 1.37 (6H, s, H-4'', 5'');

149

13

150

162.2 (C, C-2'), 160.5 (C, C-5), 158.7 (C, C-9), 158.0 (C, C-4'), 139.3 (CH, C-2''), 133.0

151

(C, C-3'''), 132.5 (CH, C-6'), 122.9 (CH, C-2'''), 122.5 (C, C-3), 119.9 (C, C-1'), 113.4

152

(CH, C-1''), 109.6 (C, C-6), 108.2 (CH, C-5'), 106.1 (C, C-10), 103.9 (CH, C-3'), 91.0

153

(CH, C-8), 77.6 (C, C-3''), 56.9 (OCH3, OCH3-7, 4'), 27.9 (CH3, CH3-4'', 5''), 26.5 (CH3,

154

CH3-4'''), 25.1 (CH2, CH2-1'''), 17.8 (CH3, CH3-5'''). HR-ESI-MS: m/z 467.1703 [M+H]+

155

(calcd. for C26H27O8 467.1700).

C NMR (CD3OD, 100 MHz) δ: 184.1 (C=O, C-4), 164.7 (C, C-7), 163.9 (C, C-2),

156

Artoheterophyllin I (5). Yellow powder: C26H30O7; UV (MeOH) 266 nm; 1H NMR

157

(Acetone-d6, 400 MHz) δ: 13.89 (1H, s, OH-5), 9.21 (2H, s, OH-2', 4'), 7.16 (1H, d, J =

158

8.4 Hz, H-6'), 6.67 (1H, dd, J = 16.4, 7.2 Hz, H-2''), 6.55 (1H, d, J = 16.4 Hz, H-1''), 6.52

159

(1H, s, H-8), 6.46 (1H, d, J = 2.4 Hz, H-3'), 6.44 (1H, dd, J = 8.4, 2.4 Hz, H-5'), 3.92 (3H,

160

s, OCH3), 2.45 (2H, m, H-1'''), 2.40 (1H, m, H-3''), 2.09, 1.05 (6H, s, H-4''', 5'''), 1.61 (2H,

161

m, H-2'''), 1.07, 1.05 (6H, d, J = 6.0 Hz, H-4'', 5'');

13

C NMR (Acetone-d6, 100 MHz) δ:


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162

184.2 (C=O, C-4), 164.5 (C, C-7), 163.1 (C, C-2), 162.1 (C, C-2'), 160.5 (C, C-5), 158.1

163

(C, C-9), 157.8 (C, C-4'), 142.8 (CH, C-2''), 132.7 (CH, C-6'), 123.7 (C, C-3), 117.6 (CH,

164

C-1''), 113.5 (C, C-1'), 110.4 (C, C-6), 108.7 (CH, C-5'), 106.2 (C, C-10), 104.5 (CH, C-

165

3'), 91.1 (CH, C-8), 70.8 (C, C-3'''), 57.2 (CH3, OCH3), 43.6 (CH2, C-2'''), 34.5 (CH, C-

166

3''), 31.2, 29.9 (CH3, C-4''', 5'''), 23.7 (CH3, CH3-4'', 5''), 21.8 (CH2, C-1'''). HR-ESI-MS:

167

m/z 455.2067 [M+H]+ (calcd. for C26H31O7 455.2064).

168

Artoheterophyllin J (6). Pale yellow powder: C16H14O7; [α]20D -200.0 (c 0.002, CHCl3);

169

UV (MeOH) 236, 288 nm; CD (MeOH, c 0.0009): [θ]276 +3566; IR (KBr) νmax 3341,

170

1612, 1594, 1469, 1279, 1152cm-1; 1H NMR (CD3OD, 400 MHz) δ: 5.97 (2H, s, H-3', 5'),

171

5.874 (1H, d, J = 2.8 Hz, H-8), 5.868 (1H, d, J = 2.8 Hz, H-6), 5.84 (1H, dd, J = 14.0, 2.8

172

Hz, H-2), 3.93 (1H, dd, J = 17.2, 14.0 Hz, H-3ax), 3.71 (3H, s, OCH3), 2.41 (1H, dd, J =

173

17.2, 2.8 Hz, H-3eq); 13C NMR (CD3OD, 100 MHz) δ: 199.9 (C=O, C-4), 168.1 (C, C-7),

174

166.1 (C, C-4'), 165.7 (C, C-9), 163.1 (C, C-5), 159.4 (C, C-2', 6'), 117. 9 (s, C-1'), 105.3

175

(CH, C-6), 103.4 (C, C-10), 96.9 (C, C-1'), 96.2 (CH, C-8), 94.6 (CH, C-3', 5'), 74.0 (CH,

176

C-2), 55.7 (CH3, OCH3), 41.3 (CH2, C-3). HR-ESI-MS: m/z 319.0814 [M+H]+ (calcd. for

177

C16H15O7 319.0812).

178

2-Geranyl-2',3,4',5-tetrahydroxy-trans-stilbene (7). Gray powder: C24H28O4; UV

179

(MeOH) 252, 271 nm; IR (KBr) νmax 3357, 2910, 1609, 1454, 1270, 1137, 1004 cm-1; 1H

180

NMR (Acetone-d6, 400 MHz) δ: 8.60, 8.42, 7.38 (3H, OH-5, 2', 4'), 8.15 (1H, OH-3),

181

7.38 (1H, d, J = 8.8 Hz, H-6'), 7.23 (2H, s, H-7', 8'), 6.66 (1H, d, J = 2.4 Hz, H-6), 6.44

182

(1H, d, J = 2.4 Hz, H-3'), 6.38 (1H, dd, J = 8.4, 2.4 Hz, H-5'), 6.33 (1H, d, J = 2.4 Hz, H-

183

4), 5.22 (1H, m, H-6''), 5.19 (1H, m, H-2''), 3.45 (2H, d, J = 6.4 Hz, H-1''), 2.28 (1H, d, J

184

= 7.2 Hz, H-5''), 2.25 (1H, d, J = 7.2 Hz, H-4''), 2.16 (1H, d, J = 7.2 Hz, H-4''), 2.12 (1H,



185

d, J = 7.2 Hz, H-5''), 1.67 (3H, s, H-8''), 1.66 (3H, s, H-9''), 1.62 (3H, s, H-10''); 13C NMR

186

(Acetone-d6, 100 MHz) δ: 159.1 (C, C-4'), 157.0 (C, C-2'), 156.9 (C, C-5), 156.8 (C, C-3),

187

140.1 (C, C-1), 134.6 (C, C-3''), 131.9 (C, C-7''), 128.3 (CH, C-6'), 126.1 (CH, C-2''),

188

125.6 (CH, C-7'), 125.5 (CH, C-6''), 124.5 (CH, C-8'), 118.4 (C, C-2), 117.7 (C, C-1'),

189

108.5 (CH, C-5'), 104.3 (CH, C-6), 103.8 (CH, C-3'), 102.5 (CH, C-4), 33.0 (CH2, C-5''),

190

27.3 (CH2, C-4''), 26.0 (CH3, C-8''), 24.8 (CH2, C-1''), 23.8 (CH3, C-10''), 17.8 (CH3, C-

191

9''). HR-ESI-MS: m/z 381.2063 [M+H]+ (calcd. for C24H29O4 381.2060).

192

5-Methoxy-morican M (8). Gum: C15H12O5; UV (MeOH) 244, 323 nm; 1H NMR

193

(Acetone-d6, 400 MHz) δ: 8.57 (2H, s, OH-3',5'), 7.82 (1H, s, OH-6), 7.12 (1H, s, H-4),

194

7.01 (1H, s, H-3), 7.00 (1H, s, H-7), 6.82 (2H, d, J = 2.4 Hz, H-2', 6'), 6.34 (1H, t, J = 2.4

195

Hz, H-4'), 3.89 (3H, s, OCH3);

196

155.7 (C, C-2), 150.7 (C, C-7a), 146.7 (C, C-6), 146.4 (C, C-5), 133.5 (C, C-1'), 121.6 (C,

197

C-3a), 103.7 (CH, C-2',6'), 103.5 (CH, C-4'), 103.4 (CH, C-4), 102.7 (CH, C-3), 98.5 (CH,

198

C-7), 56.9 (CH3, OCH3). HR-ESI-MS: m/z 273.0959 [M+H]+ (calcd. for C15H13O5

199

273.0758).

13

C NMR (Acetone-d6, 100 MHz) δ: 160.0 (C, C-3', 5'),

200

2,3-Dihydro-5,7-dihydroxy-2-(2-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one

201

(9). Pale yellow powder: C16H14O6; [α]20D -5.8 (c 0.0019, CHCl3); UV (MeOH) 274, 299

202

nm; CD (MeOH, c 0.0009): [θ]276 +6512; IR (KBr) νmax 3356, 2913, 1643, 1596, 1441,

203

1260, 1156 cm-1; 1H NMR (Acetone-d6, 400 MHz) δ : 12.20 (1H, s, OH-5), 9.65 (1H, s,

204

OH-7), 8.64 (1H, s, OH-2'), 7.41 (1H, d, J = 7.2 Hz, H-6'), 6.53 (1H, dd, J = 7.2, 2.4 Hz,

205

H-5'), 6.52 (1H, d, J = 2.4 Hz, H-3'), 5.97 (1H, d, J = 2.0 Hz, H-8), 5.95 (1H, d, J = 2.0

206

Hz, H-6), 5.73 (1H, dd, J = 13.2, 2.8 Hz, H-2), 3.76 (3H, s, OCH3), 3.17 (1H, dd, J = 17.2,

207

13.2 Hz, H-3ax), 2.74 (1H, dd, J = 17.2, 2.8 Hz, H-3eq);


13

C NMR (Acetone-d6, 100

Page 10 of 36

Page 11 of 36


208

MHz) δ: 197.7 (C=O, C-4), 167.4 (C, C-7), 165.4 (C, C-5), 164.8 (C, C-9), 162.0 (C, C-

209

4'), 156.3 (C, C-2'), 129.0 (CH, C-6'), 118.8 (C, C-1'), 106.1 (CH, C-5'), 103.3 (CH, C-

210

10), 102.5 (CH, C-3'), 96.9 (CH, C-6), 95.9 (CH, C-8), 75.4 (CH, C-2), 55.6 (CH3,

211

OCH3), 42.7 (CH2, C-3). HR-ESI-MS: m/z 303.0865 [M+H]+ (calcd. for C16H15O6

212

303.0863).

213

6-[(1S,2S)-1,2-dihydroxy-3-methylbutyl]-2-(2,4-dihydroxyphenyl)-5-hydroxy-7-

214

methoxy-3-(3-methyl-2-buten-1-yl)-4H-1-benzopyran-4-one

(10).

Yellow

powder:

215

C26H30O8; UV (MeOH) 260 nm; 1H NMR (CD3OD, 400 MHz) δ: 7.09 (1H, d, J = 8.4 Hz,

216

H-6'), 6.58 (1H, s, H-8), 6.42 (1H, d, J = 2.0 Hz, H-3'), 6.41 (1H, dd, J = 8.4, 2.0 Hz, H-

217

5'), 5.12 (1H, t, J = 7.6 Hz, H-2'''), 4.80 (1H, overlapped, H-1''), 4.33 (1H, dd, J = 8.8, 2.0

218

Hz, H-2''), 3.88 (3H, s, OCH3), 3.11 (2H, d, J = 6.8 Hz, H-1'''), 2.66 (1H, s, H-3''), 1.59,

219

1.40 (each 3H, s, H-4''', 5'''), 0.95, 0.89 (each 3H, d, J = 6.8 Hz, H-4'', 5'');

220

(CD3OD, 100 MHz) δ: 184.1 (C=O, C-4), 165.9 (C, C-7), 165.0 (C, C-2), 164.0 (C, C-2'),

221

162.2 (C, C-5), 159.8 (C, C-9), 158.0 (C, C-4'), 133.0 (C, C-3'''), 132.5 (CH, C-6'), 122.8

222

(CH, C-2'''), 122.8 (C, C-3), 113.3 (C, C-1'), 109.5 (C, C-6), 108.2 (CH, C-5'), 106.0 (C,

223

C-10), 103.9 (CH, C-3'), 91.4 (CH, C-8), 79.8 (CH, C-1''), 77.4 (CH, C-2''), 56.8 (OCH3,

224

OCH3-7), 31.1 (CH, C-3''), 26.0, 17.7 (CH3, CH3-4''', 5'''), 25.1 (CH2, CH2-1'''), 21.2, 15.5

225

(CH3, CH3-4''', 5'''). HR-ESI-MS: m/z 471.2013 [M+H]+ (calcd. for C26H31O8 471.2013).

13

C NMR

226

Cell Culture and Drug Treatment. MCF-7, SMMC-7721 and NCI-H460 cells were

227

cultured in Dulbecco’s Modified Eagle Medium (high glucose) and RPMI-1640,

228

respectively, plusing 10% fetal bovine serum under standard culture conditions. When the

229

cells grew to about 80% confluence, they were subcultured or treated with drugs. The

230

drugs' concentrations were based on the IC50 for 48 h treatment of specific agents on the



231

different cancer cell lines. To determine whether chemicals induced cancer cell apoptosis

232

through caspase-dependent pathways, the spectrum caspase inhibitor 50 µM broad Z-

233

VAD-fmk was applied for 1h before compounds 11 and 30 administrations. To determine

234

whether chemicals induced cancer cell apoptosis via p38, JNK and ERK pathways, their

235

inhibitors were used to pretreated 1 h before chemical treatment, including 20 µM

236

SB203580, 20 µM SP600125 and 20 µM PD98059.

237

Morphological Analysis of Apoptosis by DAPI Staining. Morphological changes of

238

apoptosis were detected by fluorescence microscopy after staining with 1ug/ml DAPI.19

239

All experiments were repeated three times.

240

Growth Inhibition Study. The proliferation of cells was assessed by using MTT [3-

241

(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay, as reported

242

previously.20 Cells were planted on 96-well microplates in 200 µL (5×103/well). After 24

243

h, the cells were treated with complete medium containing compounds (10, 25 and 50 µM)

244

for 48 h. At the end of experiments, 20 µL 5 µg/mL MTT was directly added to each well.

245

Cells were then incubated at 37 °C for 4h. Formazan was solubilized by 100 µl DMSO

246

and measured at 570 nm. All experiments were repeated three times.

247

Western Blot Analysis. Total cell lysates were denatured with sample loading buffer

248

and then subjected to SDS-PAGE for protein separation. After transferred onto

249

membranes, the proteins were probed with corresponding antibodies and detected by

250

ECL detection reagents (GE). The primary antibodies for PARP were from Cell Signaling

251

(Beverly, MA). Anti-β-actin was from Sigma. Secondary anti-mouse and rabbit were

252

from Thermo.


Page 12 of 36

Page 13 of 36


253

Statistical Analysis. Statistical analysis was done by using a two-tailed Student’s t-

254

test and p < 0.05 was considered significant. Data are expressed as the mean ± SD. All

255

assays were performed in three independent experiments.

256 257

RESULTS AND DISCUSSION

258

Identification of Compounds 1-33. In total, 33 phenolic compounds, including 25

259

flavonoids (3-6, 9-18, 19-27, 30-31), 3 stilbene derivatives (2, 7, 29), 2 simple resorcinols

260

(32-33), 2 benzofuran derivatives (8, 28), and one other type of compouds (1) were

261

isolated from the ethanol extract of woods of A. heterophyllus. Their structures were

262

identified as artoheterophyllin E (1), artoheterophyllin F (2), artoheterophyllin G (3),

263

artoheterophyllin H (4),

264

2',3,4',5-tetrahydroxy-cis-stilbene (7), 5-methoxy-morican M (8), 2,3-dihydro-5,7-

265

dihydroxy-2-(2-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one

266

1,2-dihydroxy-3-methylbutyl]-2-(2,4-dihydroxyphenyl)-5-hydroxy-7-methoxy-3-(3-

267

methyl-2-buten-1-yl)-4H-1-benzopyran-4-one (10) (Figure 1),22 artocarpin (11),23

268

cycloartocarpin (12),23 cycloartocarpesin (13),23 steppogenin (14),23 artocarpesin (15),23

269

norartocarpetin (16),23 brosimone I (17),23 isoartocarpesin (18),23 cyanomaclurin (19),23

270

artocarpanone (20),23 albanin A (21),24 artocarmin A (22),24 apigenin (23),25 artocarpetin

271

(24),23 gemichalcone A (25),24 artocarpfuranol (26),23 morin (27),26 moracin M (28),18

272

artocarbene

273

dihydroxybenzoic acid methyl ester (32),30 2,4-dihydroxy-benzaldehyde (33)31 on the

274

basis of spectral data and literature reports. Of these, except that 12 compounds (11-20,

275

24, 26) had been isolated and identified in our previous studies,23 additional other 24

(29),27

artoheterophyllin I (5), artoheterophyllin J (6), 2-geranyl-

cudraflavone

C

(30),28

hypargyflavones


(9),21

A

6-[(1S,2S)-

(31),29

2,4-


Page 14 of 36

276

compounds, including 8 new compounds, artoheterophyllin E (1), artoheterophyllin F (2),

277

artoheterophyllin

278

artoheterophyllin J (6), 2-geranyl-2',3,4',5-tetrahydroxy-cis-stilbene (7), 5-methoxy-

279

morican M (8), 2 new natural compounds, 2,3-dihydro-5,7-dihydroxy-2-(2-hydroxy-4-

280

methoxyphenyl)-4H-1-benzopyran-4-one

281

methylbutyl]-2-(2,4-dihydroxy-phenyl)-5-hydroxy-7-methoxy-3-(3-methyl-2-buten-1-yl)-

282

4H-1-benzopyran-4-one (10), were obtained and identified at this time.

G

(3),

artoheterophyllin

(9)

H

(4),

and

artoheterophyllin

I

(5),

6-[(1S,2S)-1,2-dihydroxy-3-

283

The molecular formula of 1 was deduced to be C15H12O5 by HR-ESI-MS. The 1H

284

NMR spectrum exhibited 12 proton signals, including three hydroxy signals at δH 9.28

285

(1H, s), 9.12 (1H, s), and 8.91 ppm (1H, s), three proton signals of δH 7.08 (1H, d, J = 8.4

286

Hz), 6.31 (1H, dd, J = 8.4, 2.4 Hz), and 6.18 ppm (1H, d, J = 2.4 Hz) forming one ABX

287

system, two resonance signals at δH 5.94 (1H, d, J = 2.0 Hz), 5.69 (1H, d, J = 2.0 Hz)

288

making up a tetrasubstituted phenyl ring, and three other signals at δH 5.20 (1H, m), 5.15

289

(1H, m), and 2.12 (2H, m). In the 1H-1H COSY spectrum, the signal at 2.12 (2H, m)

290

showed the correlations with signals at δH 5.20 (1H, m) and 5.15 (1H, m), which

291

indicated the fragment of -CH-CH2-CH-. Its 13C NMR spectrum revealed the presence of

292

15 carbons, including 12 aromatic carbons, 2 oxygenated alkyl carbons, and one

293

methylene. In the HMBC experiments, δH 9.12 ppm had correlations with carbons δC

294

103.6, 108.9, and 160.2 ppm, followed by proton signals δH 7.08 had corrections with

295

carbons δC 67.8, 108.9, 114.0, 155.8, and 160.2 ppm, δH 6.31 had corrections with

296

carbons δC 103.6, 114.0, and 160.2 ppm, δH 6.18 ppm had corrections with carbons δC

297

108.9, 114.0, 155.8, and 160.2 ppm, indicating the presence of structural fragment A. On

298

the other hand, the hydroxy signal of δH 9.28 ppm had correlations with carbons δC 96.0,


Page 15 of 36


299

102.4, and 158.2 ppm, δH 8.91 ppm had correlations with carbons δC 95.0 and 160.3 ppm.

300

At the same time, proton signal δH 5.69 ppm had correlations with carbons δC 96.0, 102.4,

301

155.8, 160.3 ppm, and proton signal δH 5.94 had correlations with carbons δC 95.0, 102.4,

302

160.3 ppm, supporting the presence of the structural fragment B. Furthermore, the proton

303

signal at δH 2.12 ppm exhibited the correlations with carbons δC 62.8, 67.8, 102.4, and

304

114.0 ppm, suggesting the structural fragments A and B were joined by the methylene

305

group. Thus, compound 1 was assigned as shown in Figure 1 and named as

306

artoheterophyllin E.

307

The molecular formula 2 was determined to be C24H26O4 by its HR-ESI-MS data.

308

Carefully analyzing the 1H and 13C NMR of 2, we found that it had a similar structure to

309

compound 30, except for an additional 3, 3-dimethylallyl group. Further examination of

310

the remaining 1H and

311

group that formed a 2-methyl-2-(4-methylpent-3-enyl) chromene structure on ring A [1H:

312

δ 6.71 (1H, d, J = 10.0 Hz, H-1''), 5.58 (1H, d, J = 10.0 Hz, H-2''), 5.13 (1H, m, H-7''),

313

2.14 (2H, m, H-4''), 1.71 (2H, m, H-5''), 1.64 (3H, s, H-9''), 1.57 (3H, s, H-8'') and 1.37

314

(3H, s, H-10''); 13C: δ 118.4 (C-1''), 127.8 (C- 2''), 78.57 (C-3''), 26.6 (C-10''), 41.9 (C-4''),

315

23.5 (C-5''), 125.2 (C-6''), 132.0 (C-7''), 17.7 (C-8'') and 25.9 (C-9'')]. This unit should be

316

situated at C-3 and C-4, and its placement was corroborated by the HMBC correlations

317

from H-1'' to C-3 and C-5, and from H-2'' to C-4. The configuration of C-3'' in

318

compounds can be defined by comparing the optical rotation with reported compounds.32

319

However, we can not get ideal data of optical rotation ([α]20D -0.008, c 0.0001, CHCl3)

320

for compound 2 due to the small amounts of material available. So the absolute

321

configuration for this compound was not further confirmed. Based on the above spectral

13

C NMR signals suggested that 2 contained a modified geranyl



Page 16 of 36

322

evidence, the structure of 2 was established as shown in Figure 1, and the compound was

323

given the trivial name artoheterophyllin F.

324

Compound 3 was pale yellow powder, its molecular formula determined to be

325

C20H18O7 by its HR-ESI-MS data, The 1H NMR and

13

326

similar to those of albanin A, which was also obtained from this plant, except for one of

327

the prenyl methyl signals [δH 1.52 ppm (3H, s), δC 25.9 ppm] changing into a hydroxy-

328

substituted methylene [δH 3.82 ppm (2H, s), δC 69.6 ppm]. In the HMBC experiment,

329

proton δ 3.14 showed the correlations with carbon δC 122.2 (C-3), 184.3 (C-4), 164.4

330

(C-2), 124.8 (C-2''), and 137.0 ppm (C-3''), further confirmed the hydroxy-substitutent

331

prenyl group was attached at C-3 of the C ring. The key HMBC corrections were shown

332

in the Figure 1. Therefore, 3 was determined as (Z)-2-(2,4-dihydroxyphenyl)-5,7-

333

dihydroxy-3-(4-hydroxy-3-methylbut-2-en-1-yl)-4H-chromen-4-one,

334

artoheterophyllin G.

C NMR spectra of 3 looked very

named

as

335

The molecular formula of compound 4 was determined to be C27H26O8 by its HR-ESI-

336

MS data. The 1H and 13C NMR data for 4 were similar to those of artocarpin. However,

337

the 1H NMR spectrum exhibited a spin system that could be assigned to a 3-hydroxy-3-

338

methyl-trans-but-l-enyl group [δH 6.76 (1H, d, J = 16.4 Hz), 6.68 (1H, d, J = 16.8 Hz),

339

and 1.37 ppm (6H, s)] and another methoxyl group (δH 3.90 ppm). This was supported by

340

the

341

oxygenated carbon (δC 77.6 ppm), two olefinic carbons (δC 139.3 and 113.4 ppm) to form

342

the 3-hydroxy-3-methyl-trans-but-l-enyl group, and another methoxyl group (δC 56.9

343

ppm).33 The above information implied that the 3-methyl-1-butenyl group of artocarpin

344

was replace with a 3-hydroxy-3-methyl-trans-but-l-enyl group and one hydroxy group

13

C NMR spectrum, displaying signals of two methyl groups (δC 27.9 ppm), one


Page 17 of 36


345

was replaced with a methoxyl group in 4. This was further confirmed by the HMBC

346

correlations from the olefinic proton at δH 6.76 ppm to C-5 (δC 160.5 ppm), C-7 (δC 164.7

347

ppm), and C-3'' (δC 77.6 ppm). Another methoxyl group was attached at C-5 by the

348

HMBC corrlation of the methoxy proton at δH 3.90 ppm with C-5 (δC 160.5 ppm). Thus,

349

the structure of 4 was determined as 5,7-dimethoxy-3-(3-methylbut-2-enyl)-6-(3-

350

hydroxy-3-methyl-trans-but-1-enyl)-flavone, and named as artoheterophyllin H.

351

The molecular formula of compound 5 was determined to be C26H30O7 by its HR-ESI-

352

MS data. The 1H and 13C NMR data for 5 were similar to those of artocarpin. However,

353

the 1H NMR spectrum exhibited a spin system that could be assigned to a 3-hydroxylated

354

isoprenyl group [δH 2.45 (2H, m), 2.09, 1.05 (6H, s), 1.61 ppm (2H, m)]. This was

355

supported by the

356

and 29.9 ppm), one oxygenated carbon (δC 70.8 ppm), two methylene carbons (δC 43.6

357

and 21.8 ppm) to form the 3-hydroxylated isoprenyl group. In the HMBC spectrum, the

358

correlations from the δH 2.45 ppm (H-1''') to C-2 (δC 163.1 ppm), C-3 (δC 123.7 ppm), C-4

359

(δC 184.2 ppm), C-2''' (δC 43.6 ppm), and C-3''' (δC 70.8 ppm), and from H-2''' to C-3 (δC

360

123.7 ppm), C-1''' (δC 21.8 ppm), C-3''' (δC 70.8 ppm), C-4''' (δC 31.2 ppm) and C-5''' (δC

361

29.9 ppm) indicated that the 3-hydroxylated isoprenyl group was attached to C-3. Thus,

362

the structure of 5 was established as depicted, and named as artoheterophyllin I.

13

C NMR spectrum, displaying signals of two methyl groups (δC 31.2

363

The molecular formula of compound 6 determined to be C20H18O7 by its HR-ESI-MS

364

data. The 1H NMR spectrum of 6 showed three one-proton double doublets at δH 5.84

365

(1H, dd, J = 14.0, 2.8 Hz), 3.93 (1H, dd, J = 17.2, 14.0 Hz) and 2.41 ppm (1H, dd, J =

366

17.2, 2.8 Hz), attributed to the H-2, H-3ax and H-3eq of a flavanone,34 a two proton

367

singlet at δH 5.97 ppm (2H, s) attributed to protons in the B ring nucleus, a pair of meta-



Page 18 of 36

368

coupled doublets at δH 5.874 (1H, d, J = 2.0 Hz), 5.868 ppm (1H, d, J = 2.0 Hz)

369

assignable to H-6 and H-8, and one methoxyl signal at δH 3.71 ppm (3H, s) assigned to 5

370

or 7 position at A ring or 4' position at B ring. In the noesy experiment, the methoxyl

371

proton signal at δH 3.71 ppm exhibited correlations with protons δH 5.97 ppm (2H, s),

372

suggested that methoxyl group was attached with 4' position at B ring. According to the

373

CD data at 270-290 nm ([θ]276 +3566), the C-2 configuration can be determined as R

374

orientation of compounds 6.35 Therefore, compound 6 was determined as 5,7,2',6'-

375

tretrahydroxy-4'-methoxyflavanone, named as artoheterophyllin J.

376

The molecular formula of compound 7 was determined as C24H28O4 by HR-ESI-MS.

377

Its molecular mass was equal to that of chlorophorin.36 Examination of the 1H and

378

NMR data of these two compounds further confirmed their structural similarities, with

379

both as a tetrahydroxystilbene containing a geranyl substituent. However, in the 1H NMR

380

spectrum of compound 7, the signal for H-2 was absent. Moreover, unlike chlorophorin,

381

the observations of a pair of protons at δH 6.66 (H-6) and 6.33 ppm (H-4), and a pair of

382

no splitting of protons δH 7.23 ppm (2H, s, H-7', 8'), due to the unsymmetrical

383

substitution pattern of A ring, implied that the geranyl unit should be attached at 2 or 6

384

position in the A ring. These NMR spectroscopic properties indicated that 7 should have

385

the geranyl unit attached at C-2. Conclusive evidence for the proposed structure of 7

386

came from the HMBC correlations that were found from δH 3.45 ppm (H-1'') to C-1 (δC

387

140.1 ppm), C-2 (δC 118.4 ppm), C-3 (δC 156.9 ppm), C-2'' (δC 126.1 ppm), and C-3'' (δC

388

134.6 ppm) (Figure 1). Accordingly, compound 7 was identified as 2-geranyl-2',3,4',5-

389

tetrahydroxy-trans-stilbene.


13

C

Page 19 of 36


390

Compound 8 was obtained as brown amorphous powder. Its molecular formula was

391

determined as C15H12O5 by HR-ESI-MS. Its 1H and 13C NMR spectra were similar to 2-

392

(3,5-dihydroxyphenyl)-5,6-dihydroxybenzofuran,37 except that another signal of a

393

methoxy was present at δH 3.89 ppm. Analysis of HSQC and HMBC spectra of 8

394

suggested that the methoxy group was present at C-5. Therefore, compound 8 was

395

determined as 2-(3,5-dihydroxyphenyl)-5-methoxy-6-hydroxybenzofuran. It was found

396

that it was determined as wittifuran X by Tan et al.38 However, further analysis its

397

NMR spectrum we also found that several chemical shifts of carbons were greatly

398

different from the report by Tan et al. The chemical shifts of C-4, C-5, C-6, C-7 were δC

399

103.4, 146.7, 146.4, and 98.5 ppm, respectively. However, it was found that they were δC

400

105.7, 147.5, 144.5, and 95.8 ppm in the literature. On the other hand, we found that the

401

chemical shifts of C-4 and C-7 were δC 106.0 and 98.4 ppm respectively when hydroxy

402

group was present at C-6 in their another paper.37 This is contradictory because if the

403

structure of wittifuran X was determined as correct, the chemistry shits of C-4 should not

404

be δC 105.7 ppm due to affected by the methoxy group, it should move to the high field,

405

namely, its chemical shift should be greater different from the substituent of hydroxy

406

group, rather than similar. At the same time, the chemistry shits of C-7 should not be δC

407

95.8 ppm due to little affected by the methoxy group, its chemical shift should be similar

408

to the substituent of hydroxy group, rather than greater difference. Therefore, wittifuran

409

X determined by Tan et al. should actually be moracin J.39 Therefore, compound 8 from

410

our study was then named as 5-methoxy-morican M.

411 412

13

C

The molecular formula of compound 9 was determined as C16H14O6 by the HR-ESIMS. Carefully comparing the 1H and

13

C NMR spectra of 9 and artocarpanone, it was



Page 20 of 36

413

found that they were very similar, except for the different substitution position of

414

methoxy group. The methoxy group was present at the C-7 position of the artocarpanone,

415

while it was present at C-2' or C-4' at the B ring of 9. Analysis of HMBC spectrum of 9

416

suggested that the methoxy group was present at C-4'. According to the CD data at 270-

417

290 nm ([θ]276 +6512), the C-2 configuration can be determined as R orientation of

418

compounds 9.35 From the above analysis, the structure of 9 was determined as 2,3-

419

dihydro-5,7-dihydroxy-2-(2-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one,21

420

new natural compound.

a

The molecular formula of compound 10 was determined to be C26H30O8 by its HR-

421 422

ESI-MS data. The 1H and

13

423

However, the 1H NMR spectrum exhibited a spin system that could be assigned to a 1,2-

424

dihydroxy-3-methylbutyl group [δH 4.80 (1H, overlapped), 4.33 (1H, dd, J = 8.8, 2.0 Hz),

425

0.95 (3H, d, J = 6.8 Hz), and 0.89 ppm (3H, d, J = 6.8 Hz)]. This was supported by the

426

13

427

oxygenated carbon (δC 79.8 and 77.4 ppm), and one methine (δC 31.3 ppm). The HMBC

428

correlations between the proton at δH 4.80 ppm and the carbons C-5 (δC 162.2 ppm), C-7

429

(δC 165.9 ppm), C-6 (δC 109.5 ppm), and C-2'' (δC 77.6 ppm) confirmed that the 1,2-

430

dihydroxy-3-methylbutyl group was attached at C-6 in the A ring. Thus, the structure of

431

10 was determined

432

dihydroxy-3-methylbutyl)flavone,

433

dihydroxyphenyl)-5-hydroxy-7-methoxy-3-(3-methyl-2-buten-1-yl)-4H-1-benzopyran-4-

434

one,22 a new natural compound.

C NMR data for 10 were similar to those of artocarpin.

C NMR spectrum, displaying signals of two methyl groups (δC 26.0 and 17.7 ppm), two

as 2',4'-dihydroxy-7-dimethoxy-3-(3-methylbut-2-enyl)-6-(1,26-[(1S,2S)-1,2-dihydroxy-3-methylbutyl]-2-(2,4-


Page 21 of 36


435

Effects of Compounds 1-33 on the Growth of MCF-7, SMMC-7721 and NCI-H460

436

Cells. In order to evaluate the effects of the compounds isolated from A. heterophyllus,

437

cell viabilities were assessed via the MTT assay upon the MCF-7, SMMC-7721 and NCI-

438

H460 cancer cell lines. After 48 h treatment, compounds 5, 11, 12, 15 and 30 reduced

439

these three cell lines viable cell percentages significantly, with IC50 values lower than 25

440

µM, while compounds 4, 7, 10, 25, 29, and 31 showed moderate cytotoxic activity, with

441

IC50 values ranging from 25 to 50 µM (Table 1). The other compounds exhibited weaker

442

cytotoxic activity on these three cancer cell lines up to 50 µM (These compounds are not

443

shown in Table 1). Furthermore, compound 30 showed the strongest inhibitory effect on

444

the growth of MCF-7, SMMC-7721 and NCI-H460 Cells (IC50 = 10.81, 12.06 and 5.19

445

µM) (Table 1). Especially, the inhibitory effects on the growth of SMMC-7721 and NCI-

446

H460 cells were more potent than the positive control, 5- fluorouracil (5-FU) (IC50 =

447

17.53 and 7.31 µM) (Table 1).

448

As for flavonoids, one or two isoprenyl group substituent on the flavonoid skeleton

449

seems to increase their cytotoxic activities, especially when the substituent happening at

450

6-position of the A ring. Examples were compounds 15 and 16, 15 and 21, compounds 15

451

and 21 have one isoprenyl group substituent at 6-position and 3-position respectively,

452

while compound 16 is lack of isoprenyl group substituent at the skeleton. Cytotoxic

453

activity tested results showed that only compound 15 exhibited better cytotoxic activity,

454

suggesting that the presence of isoprenyl group at 3-position at the skeleton can not

455

improve the cytotoxic activity than those with no isoprenyl group substituent. However,

456

for those 6-isoprenyl flavonoids, the 3-isoprenyl substituent resulted in better cytotoxic

457

activity. For example, compound 30 exhibited much stronger cytotoxic activity than that



458

of compound 15 due to its additional 3-isoprenyl substituent. Moreover, cyclization of the

459

isoprenyl groups, hydroxyl group substituent, and the position and saturation of double

460

bonds of isoprenyl groups also affected cytotoxic activities. The case of compound 11

461

exhibited more potential cytotoxic activity than those of compounds 4, 5, and 10 because

462

of its lack of hydroxyl substituent at 6-isoprenyl or 3-isoprenyl group, suggesting that the

463

hydroxyl group substituent and saturation of isoprenyl group reduced the cytotoxic

464

activities. At the same time, compound 15 showed stronger cytotoxic activity than that of

465

compound 13. Meanwhile, compound 11 also exhibited better cytotoxic activity than that

466

of compounds 12 and 31 due to the cyclization of the isoprenyl groups in skeleton of the

467

latter. In addition, compound 15 exhibited stronger cytotoxic activity than that of

468

compound 18 because of the double bond at 1,2-position of the latter, while it was at 2,3-

469

position of the former.

470

Effects of Compounds 11 and 30 on the Induction of Apoptosis. Apoptosis is an

471

essential way for tissue development and homeostasis. It also works as a defense system

472

avoiding the interruption of many toxic chemicals. It is well known that anticancer

473

chemotherapies mainly exert their elimination of cancer cells via apoptotic process,

474

characteristic by a number of cellular and biochemical hallmarks, including cell

475

shrinkage, membrane blebbing, DNA chromatin fragmentation, nucleus condensation and

476

PARP cleavage.40 To detect whether the compounds 11 and 30 could induce apoptosis,

477

DAPI staining were performed. Figure 2 A showed that the indicated cell lines (including

478

NCI-H460, MCF-7 and SMMC-7721) were administrated by compounds 11 and 30 after

479

12, 24 and 48 h incubation. The apoptotic bodies were observed clearly after 24 h

480

treatment and increased more at 48 h (Figure 2A). In addition, the apoptotic effect was


Page 22 of 36

Page 23 of 36


481

validated through a significant increase of the cleaved-PARP proteins after chemicals

482

treatment with a time-dependent manner in these three cell lines (Figure 2B). All these

483

data demonstrated that compounds 11 and 30 obviously induced apoptosis in NCI-H460,

484

MCF-7 and SMMC-7721 cell lines.

485

Effects of Compounds 11 and 30 on the Caspase Related-Apoptosis. When

486

pretreated with 50 µM broad Z-VAD-fmk (spectrum caspase inhibitor) 1h before

487

compounds 11 and 30 administration, with IC50 values for 48 h, we could observe the

488

obvious increased cell number and improved cell morphology (Figure 3A&B). These

489

data supported that compounds 11 and 30 induced apoptosis through caspase-dependent

490

pathways.

491

Effects of Compounds 11 and 30 on the MAPK Pathways Related-Apoptosis. The

492

MAPK signaling pathways, including ERK, P38 and JNK MAPK pathways, are all

493

involved in affecting cell proliferation, differentiation, survival and migration.1

494

The MAPK pathway comprises several key signaling components and phosphorylation

495

events that play a role in tumorigenesis. Especially, p38 and JNK MAPK pathways

496

exhibit a complex function in cancer development. In HCC, higher JNK1 activation and

497

lower p38 MAPK activity are correlated with an increased proliferation of tumor cells.3

498

However, the increase of phosphorylated p38α have been detected in lung and breast

499

cancer.4-5 These investigations demonstrated that p38/JNK MAPK pathways have a

500

selective function depending on the different types and stages of cancers. To determine

501

whether compounds 11 and 30 induced cell apoptosis via the MAPKs pathways, 20 µM

502

JNK inhibitor (SP600125), 20 µM p38 MAPK inhibitor (SB203580) and 20 µM ERK

503

inhibitor (PD98059) were pretreated to indicated cells 1 h before compounds treatment.



504

We observed the alteration of cancer cell morphology and number after compounds 11

505

and 30 administrations at 48 h. In SMMC-7721 cell line, pretreatment with SP600125

506

will improve the cell morphology and increase the cell number (Figure 3A&B). In MCF-

507

7 cell line, pretreatment with SB203580, PD98059 and SP600125 will improve the cell

508

status obviously (Figure 3A&B). In NCI-H460 cell line, pretreatment with SB203580 or

509

PD98059 before treatment of compound 11 or 30 will elevate the cell viability,

510

respectively (Figure 3A&B). These findings provide evidence demonstrating that the

511

apoptosis-inducing effects of compounds 11 and 30 are mediated by down-regulation of

512

different MAPK pathways in different cancer cell lines separately. All above results are

513

consistent with the previous findings2-5 that MAPK pathways have a selective action on

514

different cancer cell lines.

515

In this study, eight new phenolic compounds, artoheterophyllin E-J (1-6), 4-geranyl-

516

2',3,4',5-tetrahydroxy-cis-stilbene (7),

517

compounds (9-10), 2,3-dihydro-5,7-dihydroxy-2-(2-hydroxy-4-methoxyphenyl)-4H-1-

518

benzopyran-4-one

519

dihydroxyphenyl)-5-hydroxy-7-methoxy-3-(3-methyl-2-buten-1-yl)-4H-1-benzopyran-4-

520

one, together with twenty-three known compounds (11-33), from the ethanol extract of

521

the wood of A. heterophyllus. To our knowledge, it is the first time to systematically

522

report the phenolic compounds from the wood of A. heterophyllus with their cytotoxic

523

effects on the three cell lines of MCF-7, SMMC-7721 and NCI-H460 at the same time.

524

This study demonstrates that compounds 11 and 30 from wood of A. heterophyllus

525

inhibited the growth of the three cancer cell lines by inducing caspase-related apoptosis.

526

Furthermore, the induction of apoptosis was accompanied by the activation of ERK, P38

and

5-methoxy-morican M (8), two new natural

6-[(1S,2S)-1,2-dihydroxy-3-methylbutyl]-2-(2,4-


Page 24 of 36

Page 25 of 36


527

and JNK MAPK pathways in a cell type-related manner. These data suggested that

528

compounds 11 and 30 from wood of A. heterophyllus have an anti-cancer potential and

529

can be developed as anticancer drug leads.

530 531

AUTHOR INFORMATION

532

Corresponding Author

533

*E-mail:[email protected]. Phone: +86-51085329032. Fax: +86-51085329032.

534

Funding

535

We thank the National Basic Research Program of china (973 Program, 2012CB720801),

536

the Fundamental Research Funds for the Central Universities (JUSRP11220),

537

Independent Research Program of State Key Laboratory of Food Science and Technology

538

(5812060204120150), and the grant from the National Natural Science Foundation of

539

China (No. 81202956) for the financial support.

540

Author Contributions

541

ǁ

542

Notes

543

The authors declare no competing financial interest.

Z.-P. Zheng and Y. Xu contributed equally to this work.

544 545

REFERENCES

546

(1) Wagner, E. F.; Nebreda, A. R. Signal integration by JNK and p38 MAPK pathways in

547

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Artocarpus integer and its biological activities. J. Braz. Chem. Soc. 2013, 24, 1656-1661.


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Artocarpus integrifolia distinguishes between two lymphoma lines with different

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H.; Wang, C. G.; Hu, J. F. Triterpenoids and steroids from the fruits of Melia toosendan

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Moraceae plants. 10. Cudraflavones C and D, two new prenylflavones from the root

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bark of Cudrania tricuspidata (Carr.) Bur. Heterocycles 1990, 31, 1339-1344.

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isoprenylated flavones and stilbene derivative from Artocarpus hypargyreus. Chem.

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Biodivers. 2012, 9, 394-402.

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orthogonality in the deprotection of 2,4-di-protected aromatic ethers employing solid-

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supported acids. Tetrahedron 2009, 65, 4316-4325.

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designed to form polymetallic complexes. Tetrahedron 2012, 68, 133-144.

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Lawrence, J. A.; Eldridge, G. R. Antibacterial chromene and chromane stilbenoids from



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Hymenocardia acida. Phytochemistry 2014, 98, 216-222.

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K.; Ahn, J. S. Protein tyrosine phosphatase-1B inhibitory activity of isoprenylated

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Suksamrarn, A. Bioactive flavonoids of the flowers of Butea monosperma. Chem. Pharm.

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(36) Christensen, L. P.; Lam, J.; Sigsgaard, T. A novel stilbene from the wood of

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Chlorophora excelsa. Phytochemistry 1988, 27, 3014-3016.

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(37) Tan, Y.; Liu, C.; Chen, R. 2-Arylbenzofuran derivatives from Morus wittiorum.

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(38) Tan, Y.; Yang, Y.; Zhang, T.; Chen, R. Y.; Yu, D. Q. Bioactive 2-arylbenzofuran

653

derivatives from Morus wittiorum. Fitoterapia 2010, 81, 742-746.

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658 659 660


Page 30 of 36

Page 31 of 36


661

FIGURE CAPTIONS

662

Figure 1. The key HMBC and Noesy of the compounds 1-10.

663

Figure 2. Compounds 11 and 30 induce apoptosis in NCI-H460, MCF-7 and SMMC-

664

7721 cancer cell lines. (A) After the drug administration with indicated concentration, the

665

cells were stained by DAPI and visualized by fluorescent microscope at 12, 24 and 48 h.

666

(B) Western blot analysis was used to estimate PARP cleavage after the drug

667

administration for 48 h. Results are representative of three independent experiments (n =

668

3).

669

Figure 3. Effects of compounds 11 and 30 on the three MAPK pathways inhibitors.

670

Morphological and quantitative changes in NCI-H460, MCF-7 and SMMC-7721 cells

671

either in control (untreated), treated with compound 11 (10 µM for NCI-H460, 10 µM for

672

MCF-7 and 15 µM for SMMC-7721) and 30 (5 µM for NCI-H460, 10 µM for MCF-7 and

673

12 µM for SMMC-7721) for 48 h and pretreated with 20 µM SB203580, 20µM PD98059,

674

20µM SP600125 or 50 µM Z-VAD-fmk for 1 h before compounds administration.

675

Results are representative of three independent experiments (n = 3).

676 677 678 679 680 681 682 683



684

TABLES

685

Table 1. Inhibitory Effect of the Isolated Compounds against Different Cancer Cell Lines.

686

Cancer Cell Line IC50 ± SDa (µM) Compounds MCF-7 SMMC-7721 NCI-H460 > 50 25 ~ 50 25 ~ 50 4 26.41 ± 0.95 25.28 ± 1.21 15.82 ± 0.61 5 25 ~ 50 25 ~ 50 16.66 ± 0.87 7 > 50 ≈ 50 25 ~ 50 10 11.3 ± 0.51 15.85 ± 0.79 11.01 ± 0.81 11 20.68 ± 1.01 23.7 ± 0.86 20.72 ± 0.63 12 25 ~ 50 25 ~ 50 25 ~ 50 25 25 ~ 50 25 ~ 50 25 ~ 50 29 10.81 ± 0.67 12.06 ± 0.75 5.19 ± 0.14 30 25 ~ 50 25 ~ 50 ≈ 50 31 1.11 ± 0.13 17.53 ± 0.87 7.31 ± 0.33 5-FUb a Values are means ± SD (n = 3). bPositive control.

687 688 689 690 691 692 693 694 695 696 697 698 699 700


Page 32 of 36

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701


TOC Graphic

702



178x137mm (300 x 300 DPI)


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96x117mm (300 x 300 DPI)



48x29mm (300 x 300 DPI)


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Characterization of Antiproliferative Activity Constituents from

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