Molecular Characterization and Bioactivity of Coumarin Derivatives

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Bioactive Constituents, Metabolites, and Functions

Molecular Characterization and Bioactivity of Coumarin Derivatives from the Fruits of Cucumis bisexualis Qin-Ge Ma, Rong-Rui Wei, Ming Yang, Xiao-Ying Huang, Fang Wang, Zhi-Pei Sang, Wen-Min Liu, and Qing Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00976 • Publication Date (Web): 18 May 2018 Downloaded from http://pubs.acs.org on May 18, 2018

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Journal of Agricultural and Food Chemistry

1

Molecular Characterization and Bioactivity of Coumarin Derivatives

2

from the Fruits of Cucumis bisexualis

3 4

Qin-Ge Maa,b,*, Rong-Rui Weia,*, Ming Yanga,*, Xiao-Ying Huanga, Fang Wanga, Zhi-Pei Sangb,

5

Wen-Min Liub, Qing Yub

6 7

a

8

Reduction Pharmaceutical Equipment, Key Laboratory of Modern Preparation of TCM of

9

Ministry of Education, Research Center of Natural Resources of Chinese Medicinal Materials and

State Key Laboratory of Innovative Drugs and High Efficiency Energy Saving and Consumption

10

Ethnic Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004 China

11

b

12

473061 China

College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang

13 14

*Corresponding Author:

15

Dr. Qinge Ma; Dr. Rongrui Wei; Prof. Ming Yang

16

E-mail: [email protected]

17 18 19

ABSTRACT

20

Cucumis bisexualis (Cucurbitaceae) is known as "mapao egg" or "muskmelon egg", which

21

has been widely used as a wild melon in Chinese folk. Nine new coumarin derivatives (1-9),

22

named 7-hydroxy-3-(4',6'-dihydroxy-5'-isopropyl-3'',3''-dimethyl-2H-chromen)-6-prenyl-2H-chro-

23

men-2-one (1), 7-hydroxy-3-(5'-prenyl-3'',3''-dimethyl-2H-chromen)-6-prenyl-2H-chromen-2-one

24

(2), 3-(6'-hydroxy-5'-prenyl-3'',3''-dimethyl-2H-chromen)-6-prenyl-2H-chromen-2-one (3), 3-(5'-

25

ethyl-3'',3''-dimethyl-2H-chromen)-6-prenyl-2H-chromen-2-one (4), 3-(4',6'-dihydroxy-5'-dimeth-

26

ylallyl-3'',3''-dimethyl-2H-chromen)-6-prenyl-2H-chromen-2-one (5), 3-[4',6'-dihydroxy-5'-(2-pro-

27

penyl)-3'',3''-dimethyl-2H-chromen]-14,15-dimethyl-pyrano-chromen-2-one (6), 3-(6'-dihydroxy-

28

5'-isopropanol-3'',3''-dimethyl-2H-chromen)-14,15-dimethyl-pyrano-chromen-2-one (7), 3-(5'-iso1

ACS Paragon Plus Environment


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pentenol-3'',3''-dimethyl-2H-chromen)-14,15-dimethyl-pyrano-chromen-2-one (8), 3-(4',6'-dihydr-

30

oxy-5'-prenyl-3'',3''-dimethyl-2H-chromen)-14,15-dimethyl-pyrano-chromen-2-one (9), together

31

with twelve known compounds (10-21), were isolated and identified by spectroscopic analysis and

32

references from the active site (EtOAc soluble fraction) of the fruits of C. bisexualis for the first

33

time.

34

hepatoprotective activities for the first time. Compounds 1, 3, 5, 6, 7, and 9 showed anti-AChE

35

activities with IC50 values ranging from 11.23 to 89.69 µM, and compounds 2, 4, 12, 15, 17, 18,

36

and 19 (10µM) exhibited moderate hepatoprotective activities. These findings shed much light on

37

a better understanding of the anti-AChE and hepatoprotective effects of these coumarin

38

derivatives and provided new insights into developing better anti-AChE and hepatoprotective

39

drugs in the future.

Compounds

(1-21)

were

evaluated

for

anti-acetylcholinesterase

(AChE)

and

40 41 42

KEYWORDS Cucumis bisexualis; coumarin derivative; anti-AChE; hepatoprotective

43 44 45

■ Introduction

46

Cucumis bisexualis A.M. Lu ＆ G.C. Wang is an annual creeping herbaceous plant, which

47

belongs to the Cucurbitaceae family. It is a Chinese endemic plant, and has a better ability to grow

48

in hillside, field, and roadsides. It mainly distributes in Henan, Shandong, Anhui, and Jiangsu

49

provinces in China.1 The fruit of C. bisexualis is known as "mapao egg" or "muskmelon egg", and

50

it has been widely served as a wild melon in Chinese folk.2 Based on previous pharmacological

51

investigations of C. bisexualis, it was found that there had no reports about traditional application,

52

anti-AChE, and hepatoprotective activities. Moreover, we found that there were also no reports

53

about previous phytochemical analysis of the fruits of C. bisexualis after consulting a large

54

number of references. There was only one paper on the bioactivity of the fruits of C. bisexualis, in

55

which it was reported that its extract could reduce fasting blood glucose in diabetic mice, increase

56

the activities of Mn-SOD and GSH-Px in serum, reduce the content of MDA in serum, and 2


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enhance the antioxidant capacity of type 2 diabetic mice,3 which prompted us to investigate its

58

further chemical constituents. In this work, we carried out a bioassay-guided investigation of C.

59

bisexualis in order to evaluate its anti-AChE and hepatoprotective activities. As a result, nine new

60

compounds (1-9), along with twelve known compounds (10-21), were isolated from the fruits of C.

61

bisexualis for the first time. All the compounds (1-21) were identified as coumarin derivatives by

62

extensive UV, IR, MS, NMR spectroscopic data and comparison with their references. Meanwhile,

63

these compounds (1-21) were evaluated for their anti-AChE and hepatoprotective activities for the

64

first time. These research results may guide the search for new natural products with anti-AChE

65

and hepatoprotective attributes.

66

■ Materials and methods

67

General experimental procedures. The infrared (IR) spectroscopic data were acquired from

68

a Nicolet 5700 FT-IR spectrometer with KBr pellets (Shanghai Xiangrun Industry Co. Ltd.,

69

Shanghai, China). The ultraviolet (UV) spectra were recorded on an Australia GBC UV-916

70

spectrophotometer (GBC Scientific Equipment Pty. Ltd., Braeside, Australia). The nuclear

71

magnetic resonance (NMR) spectra were recorded by a Bruker Avance III 400 spectrometer with

72

TMS as internal standard at 25 °C (Bruker Corporation, Madison, America). The high-resolution

73

electrospray ionization mass spectrometry (HR-ESI-MS) was performed with an Agilent 1100

74

series LC/MSD ion trap mass spectrometer (Agilent Technologies, California, America), and

75

electrospray ionization mass spectrometry (ESI-MS) spectra were recorded on a LTQ Orbitrap XL

76

spectrometer

77

prep-high-performance liquid chromatography (prep-HPLC) separation was performed on a

78

Shimadzu LC-6AD instrument with a SPD-20A detector and an YMC-Pack ODS-A column (250

79

× 20 mm, 5 µm) (Shimadzu Corporation, Japan), and the high-performance liquid chromatography

80

(HPLC) data were recorded on an Agilent 1200 series with a DIKMA (4.6 × 250 mm) analytical

81

column packed with C18 (5 µm) (Agilent Technologies, California, America).4 The column

82

chromatography (CC) was subjected to silica gel (100-200 or 200-300 mesh, Qingdao Marine

83

Chemical Inc., Qingdao, China) and Sephadex LH-20 (Amersham Pharmacia Biotech Co., Ltd.,

84

Tokyo, Japan). The thin-layer chromatography (TLC) detections were applyed to silica gel GF254

(Thermo

Fisher

Scientific,

Waltham,

MA,

3


USA),

respectively.

The


85

plates (Qingdao Marine Chemical Inc., Qingdao, China) and the spots were observed under UV

86

light (254 or 365 nm) or by spraying with 10% H2SO4 in 95% EtOH followed by heating.5

87

Plant material. The fruits of C. bisexualis were collected from Nanyang city of Henan

88

Province of China in september 2016 and were authenticated by Dr. Rongrui Wei of Jiangxi

89

University of Traditional Chinese Medicine. A voucher specimen (NO. XMP-201609) has been

90

deposited in Nanyang Normal University, Nanyang 473061, China.

91

Extraction and isolation. The air-dried fruits of C. bisexualis (15.5 kg) were powdered and

92

extracted with 90% EtOH (48 L) under reflux 3 times at room temperature. The filtrates were

93

combined and concentrated under reduced pressure until elimination of ethanol to obtain a dark

94

black crude extract (1.6 kg). The extract was suspended in distilled water (15.0 L) and partitioned

95

using petroleum ether (3 × 15.0 L), EtOAc (3 × 15.0 L), and n-BuOH (3 × 15.0 L), consecutively,

96

yielding petroleum ether (98.6 g), EtOAc (235.5 g), and n-BuOH (305.8 g) layers, respectively.

97

According to the screening results of bioactivity-guided investigation, the EtOAc layer exhibited

98

anti-AChE and hepatoprotective activities.

99

The EtOAc layer was chromatographed over silica gel (100-200 mesh) eluting with a

100

gradient elution (n-hexane/EtOAc = 12:1 to 3:1, v/v) to yield four fractions: A (29.6 g), B (59.5 g),

101

C (72.8 g), and D (19.5 g). The fraction B was chromatographed through a silica gel (100-200

102

mesh) column using a gradient elution (petroleum ether/EtOAc = 15:1 to 4:1, v/v) to afford three

103

sub-fractions: B1 (12.5 g), B2 (23.0 g), and B3 (9.8 g). The sub-fraction B1 was separated over

104

Sephadex LH-20 (95% MeOH in H2O), then separated by prep-HPLC (detection at 220 nm, 6

105

mL/min), successively, yielding 2 (9.05 mg), 4 (10.28 mg), 12 (11.20 mg), 13 (9.56 mg), 14

106

(10.35 mg), and 15 (13.47 mg). Meanwhile, the sub-fraction B2 was separated over Sephadex

107

LH-20 (95% MeOH in H2O) and prep-HPLC (detection at 220 nm, 6 mL/min), successively,

108

yielding 1 (8.68 mg), 3 (11.45 mg), 5 (9.68 mg), 17 (12.75 mg), 18 (13.20 mg), and 19 (14.32

109

mg).

110

In the same way, the fraction C was submitted to silica gel column (100-200 mesh) eluting

111

with a gradient elution (petroleum ether/EtOAc = 10:1 to 3:1, v/v) to yield four fractions: C1

112

(12.5 g), C2 (26.3 g), C3 (20.4 g), and C4 (10.6 g). The sub-fraction C2 was separated over

113

Sephadex LH-20 (90% MeOH in H2O), then separated by prep-HPLC (detection at 220 nm, 6 4


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114

mL/min), successively, yielding 6 (10.34 mg), 7 (13.62 mg), 16 (12.81 mg), and 21 (14.05 mg).

115

The sub-fraction C3 was fractionated on Sephadex LH-20 (90% MeOH in H2O), then separated by

116

prep-HPLC (detection at 220 nm, 6 mL/min), successively, yielding 8 (8.93 mg), 9 (12.37 mg), 10

117

(13.54 mg), 11 (12.05 mg), and 20 (13.76 mg). The flow chart for extraction and separation of

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compounds (1-21) are shown in Figure 1.

119

7-hydroxy-3-(4',6'-dihydroxy-5'-isopropyl-3'',3''-dimethyl-2H-chromen)-6-prenyl-2H-chrome

120

n-2-one (1): colorless powder, HR-ESI-MS: m/z 485.2531 [M+Na]+ (calcd. for C28H30O6Na,

121

485.2532); UV (MeOH) λmax: 220, 258, 285, and 355 nm; IR νmax: 3346, 1681, 1626, and 1384

122

cm-1; 1H NMR (Acetone-d6, 400 MHz) and 13C NMR (Acetone-d6, 100MHz) data see Table 1.

123

7-hydroxy-3-(5'-prenyl-3'',3''-dimethyl-2H-chromen)-6-prenyl-2H-chromen-2-one

(2):

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colorless powder, HR-ESI-MS: m/z 479.1563 [M+Na]+ (calcd. for C30H32O4Na, 479.1566); UV

125

(MeOH) λmax: 221, 257, 285, and 354 nm; IR νmax: 3345, 1680, 1627, 1385 cm-1; 1H NMR

126

(Acetone-d6, 400 MHz) and 13C NMR (Acetone-d6, 100MHz) data see Table 1.

127

3-(6'-hydroxy-5'-prenyl-3'',3''-dimethyl-2H-chromen)-6-prenyl-2H-chromen-2-one

(3):

128


129

(MeOH) λmax: 221, 258, 285, and 355 nm; IR νmax: 3345, 1680, 1627, and 1384 cm-1; 1H NMR

130


131

3-(5'-ethyl-3'',3''-dimethyl-2H-chromen)-6-prenyl-2H-chromen-2-one (4): colorless powder,

132

HR-ESI-MS: m/z 423.40128 [M+Na]+ (calcd. for C27H28O3Na, 423.4025); UV (MeOH) λmax: 220,

133

257, 286, and 355 nm; IR νmax: 3349, 1681, 1628, 1462, and 1385 cm-1; 1H NMR (Acetone-d6, 400

134

MHz) and 13C NMR (Acetone-d6, 100MHz) data see Table 2.

135

3-(4',6'-dihydroxy-5'-dimethylallyl-3'',3''-dimethyl-2H-chromen)-6-prenyl-2H-chromen-2-one

136

(5): colorless powder, HR-ESI-MS: m/z 495.3219 [M+Na]+ (calcd. for C30H32O5Na, 495.3212);

137

UV (MeOH) λmax: 221, 257, 285, and 355 nm; IR νmax: 3346, 1681, 1628, and 1385 cm-1; 1H NMR

138


139

3-[4',6'-dihydroxy-5'-(2-propenyl)-3'',3''-dimethyl-2H-chromen]-14,15-dimethyl-pyranochro-

140

men-2-one (6): colorless powder, HR-ESI-MS: m/z 481.4013 [M+Na]+ (calcd. for C28H26O6Na,

141


142

cm-1; 1H NMR (Acetone-d6, 400 MHz) and 13C NMR (Acetone-d6, 100MHz) data see Table 2. 5



143

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3-(6'-dihydroxy-5'-isopropanol-3'',3''-dimethyl-2H-chromen)-14,15-dimethyl-pyrano-chrome

144

n-2-one (7): colorless powder, HR-ESI-MS: m/z 483.3528 [M+Na]+ (calcd. for C28H28O6Na,

145


146


147

3-(5'-isopentenol-3'',3''-dimethyl-2H-chromen)-14,15-dimethyl-pyrano-chromen-2-one

(8):

148


149

(MeOH) λmax: 221, 258, 286, and 354 nm; IR νmax: 3347, 1681, 1628, and 1384 cm-1; 1H NMR

150


151

3-(4',6'-dihydroxy-5'-prenyl-3'',3''-dimethyl-2H-chromen)-14,15-dimethyl-pyrano-chromen-2

152

-one (9): colorless powder, HR-ESI-MS: m/z 509.2074 [M+Na]+ (calcd. for C30H30O6Na,

153


154


155

Anti-AChE activity assay. The compounds (1-21) were assayed for their anti-AChE

156

activities by an acetylthiocholine iodide substrate-based colorimetric method.6 The whole brains

157

of mice were homogenized in a hand homogenizer with 10 volumes of homogenization buffer

158

[400 mM NaCl and 12.5 mM sodium phosphate buffer (pH 7.0)], and obtained the supernatant as

159

the enzyme for a future assay after centrifuging at 1000 g for 10 min at 4 °C. The compounds

160

(1-21) were dissolved in buffer A [100 mM sodium phosphate buffer (pH 8.0)] and diluted to

161

various concentrations in buffer A. An aliquot of the isolate solution diluted in buffer A (1.5 mL)

162

was mixed with buffer A (2.6 mL), an acetylthiocholine iodide solution (20 µL, 75 mM), and

163

buffered Ellman’s reagent [100 µL, 10 mM DTNB (dithiobisnitrobenzoic acid) and 15 mM

164

sodium bicarbonate], then reacted for 30 min at room temperature. The absorbance at 412 nm was

165

immediately measured after the enzyme source (400 µL) had been added to the reaction mixtures

166

(UV-1700 PharmaSpec, Shimadzu Co. Ltd.) and recorded data at 30 s intervals for 5 min. The

167

enzyme inhibition dose-response curve was used to calculate the half-inhibition rate against AChE

168

activity (IC50).

169

Hepatoprotective assay. The compounds (1-21) were evaluated for their hepatoprotective

170

activities against D-galactosamine induced toxicity in HL-7702 cells by using a MTT colorimetric

171

method.7 The HL-7702 cell lines were cultured in Dulbecco's modified eagle medium (DMEM) 6


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supplemented with 3% fetal calf serum, 100 units/mL penicillin, and 100 units/mL streptomycin in

173

5% CO2 and incubated at 37 °C, which were placed in a 96-well microplate and precultured for 24

174

h. The cultured cells were measured for cytotoxic effects which exposed to 40 mM

175

D-galactosamine after 24 h.8 Lastly, the medium was replaced for the serum-free medium (0.5

176

mg/mL MTT) for 3.5 h incubation. After removing of the medium, DMSO (150 µL/well) was

177

added into the microplate, and the formazan crystals were redissolved. The optical density (OD)

178

was recorded on a microplate reader at a wavelength of 492 nm, and the inhibition was calculated

179

as inhibition (%) = [(OD(sample) - OD(control))/(OD(normal) - OD(control))] × 100.9

180

■ Results and discussion

181

Structure elucidation of new compounds. Compound 1 was isolated as a colorless powder

182

whose molecular formula was established as C28H30O6 from the HR-ESI-MS ion peak at m/z

183

485.2531 [M+Na]+ (calcd. for C28H30O6Na, 485.2532), indicating fourteen degrees of unsaturation.

184

The UV spectrum of compound 1 showed the absorptions at λmax 220, 258, 285, and 355 nm and

185

resembled that of 3-arylcoumarin.10 The IR spectrum of compound 1 suggested the presence of

186

hydroxyl (3346 cm-1), carbonyl (1681 cm-1), aromatic ring (1626 cm-1), and methyl (1384 cm-1)

187

functionalities.

188

The 1H NMR data (Table 1) of compound 1 showed signals for a prenyl group (δH 3.34, 2H,

189

d, J = 7.2 Hz, H-11; δH 5.27, 1H, t, J = 7.2, 1.5 Hz, H-12; δH 1.77, 3H, s, 14-CH3; δH 1.65, 3H, s,

190

15-CH3), a 3′′,3′′-dimethyl-pyran ring (δH 6.60, 1H, d, J = 10.0 Hz, H-1′′; δH 5.68, 1H, d, J = 10.0

191

Hz, H-2′′; δH 1.46, 6H, s, 4′′/5′′-CH3), and a isopropyl group (δH 2.68, 1H, m, H-7′; δH 1.14, 6H, s,

192

8′/9′-CH3). Moreover, there were three singlets (δH 7.53, 1H, s, H-4; δH 7.12, 1H, s, H-5; δH 6.75,

193

1H, s, H-8) and a typical carbonyl carbon (δC 160.8, C-2) from the NMR data of compound 1

194

confirmed the existence of the 3-arylcoumarin skeleton.10 All of the above fragments were

195

connected by the HMBC correlations of H-4/C-2; H-4/C-5; H-4/C-1′; H-5/C-11; H-8/C-10;

196

H-12/C-6; H-7′/C-4′; H-7′/C-6′; H-1′′/C-4′; H-1′′/C-3′′ (Figure 2), the 2D-NOESY correlations of

197

H-12/14-CH3; H-12/15-CH3; 8′-CH3/9′-CH3; H-2′′/5′′-CH3 (Figure 2), and the 1H-1H COSY

198

correlations of H-11/H-12; H-1′′/H-2′′ (Figure 2). Therefore, compound 1 was determined as

199

7-hydroxy-3-(4',6'-dihydroxy-5'-isopropyl-3'',3''-dimethyl-2H-chromen)-6-prenyl-2H-chromen-2-

7



200

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one.

201

Compound 2 was obtained as a colorless powder. It showed a molecular formula of C30H32O4

202

based on the HR-ESI-MS ion at m/z 479.1563 [M+Na]+ (calcd. for C30H32O4Na, 479.1566)

203

corresponding to fifteen degrees of unsaturation. The UV spectrum of compound 1 showed the

204

absorptions

205

characteristic absorption peaks at 3345, 1680, 1627, 1385 cm-1, which indicated the characteristic

206

absorption peaks of 3-arylcoumarin.10 Compound 2 was concluded to be an analogue of

207

compound 1 according to its spectral data of 1H NMR and

208

between compound 2 and compound 1 which showed two singlets (δH 6.81, 1H, s, H-4′; δH 6.94,

209

1H, s, H-6′) and 5′-prenyl (δH 3.32, 2H, d, J = 7.2 Hz, H-7′; δH 5.24, 1H, t, J = 7.2, 1.5 Hz, H-8′;

210

δH 1.76, 3H, s, 10′-CH3; δH 1.66, 3H, s, 11′-CH3) in compound 2. The fragments of compound 2

211

were connected by the HMBC correlations of H-4/C-2; H-4/C-5; H-4/C-1′; H-5/C-11; H-8/C-10;

212

H-12/C-6; H-4′/C-7′; H-7′/C-9′; H-8′/C-5′; H-1′′/C-4′; H-1′′/C-3′′ (Figure 2), the 2D-NOESY

213

correlations of H-12/14-CH3; H-12/15-CH3; H-8′/10′-CH3; H-2′′/5′′-CH3 (Figure 2), and the 1H-1H

214

COSY correlations of H-11/H-12; H-7′/H-8′; H-1′′/H-2′′ (Figure 2). Consequently, compound 2

215

was elucidated as 7-hydroxy-3-(5'-prenyl-3'',3''-dimethyl-2H-chromen)-6-prenyl-2H-chromen-2-

216

one.

at

λmax

221,

257,

285,

354

nm

and

13

its

IR

spectrum

showed

C NMR (Table 1). The differences

217

Compound 3 was obtained as a colorless powder. Its molecular formula was determined as

218

C30H32O4 by analysis of a HR-ESI-MS ion at m/z 479.1423 [M+Na]+ (calcd. for C30H32O4Na,

219

479.1427). The UV and IR spectral data of compound 3 were similar to those of compound 2,

220

which showed the absorptions at λmax 221, 258, 285, 355 nm and indicated the existence of

221

hydroxyl (3345 cm-1), carbonyl (1680 cm-1), aromatic ring (1627 cm-1), methyl (1384 cm-1)

222

functional groups, separately. It can be concluded that compound 3 was an analogue of compound

223

2.10 In the 1H NMR spectrum of compound 3 (Table 1), a typical ABX system (δH 7.16, 1H, d, J =

224

2.0 Hz, H-5; δH 6.98, 1H, dd, J = 8.0, 2.0Hz, H-7; δH 6. 85, 1H, d, J = 8.0 Hz, H-8) revealed that a

225

6,9,10-trisubstituted phenyl moiety and a singlet (δH 6.79, 1H, s, H-4′) were in compound 3. The

226

structure of compound 3 was determined by the HMBC correlations of H-4/C-2; H-4/C-5;

227

H-4/C-1′; H-5/C-11; H-7/C-11; H-8/C-10; H-4′/C-7′; H-7′/C-9′; H-8′/C-5′; H-1′′/C-4′; H-1′′/C-3′′

228

(Figure 2), the 2D-NOESY correlations of H-12/14-CH3; H-12/15-CH3; H-2′′/5′′-CH3; 8


Page 9 of 24


229

H-8′/10′-CH3 (Figure 2), and the 1H-1H COSY correlations of H-7/H-8; H-11/H-12; H-7′/H-8′;

230

H-1′′/H-2′′ (Figure 2). Thus, compound 3 was elucidated as 3-(6'-hydroxy-5'-prenyl-3'',3''-dimeth-

231

yl-2H-chromen)-6-prenyl-2H-chromen-2-one.

232

Compound 4 was isolated as a colorless powder. The molecular formula of compound 4 was

233

found to be C27H28O3 by the HR-ESI-MS ion at m/z 423.4028 [M+Na]+ (calcd. for C30H32O4Na,

234

423.4025) with fourteen degrees of unsaturation. Compound 4 was concluded to be an analogue of

235

compound 3; its UV spectrum showed absorbance at λmax 220, 257, 286, and 355 nm and its IR

236

spectrum displayed absorption peaks at 3349, 1681, 1628, 1462, 1385 cm-1.10 The 1H and

237

NMR spectral data of (Table 2) were similar to those of compound 3 except for two singlets (δH

238

6.82, 1H, s, H-4′; δH 6.93, 1H, s, H-6′) and 5′-ethyl (δH 2.68, 2H, m, H-7′; δH 1.19, 3H, s, 8′-CH3)

239

in compound 4. The structure of compound 4 was confirmed by the key HMBC correlation of

240

H-4/C-2; H-4/C-5; H-4/C-1′; H-5/C-11; H-7/C-11; H-8/C-10; H-4′/C-7′; H-6′/C-7′; H-1′′/C-4′;

241

H-1′′/C-3′′ (Figure 2), the 2D-NOESY correlations of H-12/14-CH3; H-12/15-CH3; H-2′′/5′′-CH3;

242

H-8′/10′-CH3 (Figure 2), and the 1H-1H COSY correlations of H-7/H-8; H-11/H-12; H-1′′/H-2′′

243

(Figure 2). Therefore, compound 4 was determined to be 3-(5'-ethyl-3'',3''-dimethyl-2H-chromen)-

244

6-prenyl-2H-chromen-2-one.

13

C

245

Compound 5 was isolated as a colorless powder. The molecular formula of compound 5 was

246

assigned as C30H32O5 from the HR-ESI-MS signal of the sodium adduct ion at m/z 495.3219

247

[M+Na]+ (calcd. for C30H32O5Na, 495.3212). Its UV (MeOH) λmax: 221, 257, 285, 355 nm and IR

248

νmax: 3346, 1681, 1628, 1385 cm-1 indicated compound 5 was an analogue of compound 3.10 The

249

1

250

δH 6.98, 1H, dd, J = 8.0, 2.0 Hz, H-7; δH 6.87, 1H, d, J = 8.0 Hz, H-8) revealing that a

251

6,9,10-trisubstituted phenyl moiety and a typical dimethylallyl (δH 6.31, 1H, dd, J = 17.5, 10.7 Hz,

252

H-8′; δH 5.02, 1H, dd, J = 17.5, 1.3 Hz, H-9′a; δH 5.00, 1H, dd, J = 10.7, 1.3 Hz, H-9′b; δH 1.53,

253

6H, s, 10′/11′-CH3) in compound 5.11 The fragments of compound 5 were connected by HMBC

254

correlation of H-4/C-2; H-4/C-5; H-4/C-1′; H-5/C-11; H-7/C-11; H-8/C-10; H-9′/C-7′; H-1′′/C-4′;

255

H-1′′/C-3′′ (Figure 2), the 2D-NOESY correlations of H-12/14-CH3; H-12/15-CH3; H-2′′/5′′-CH3;

256

H-9′/10′-CH3 (Figure 2), and the 1H-1H COSY correlations of H-7/H-8; H-11/H-12; H-1′′/H-2′′;

257

H-8′/H-9′ (Figure 2). Thus, compound 5 was determined to be 3-(4',6'-dihydroxy-5'-dimethylallyl-

H NMR spectra showed the presence of a typical ABX system (δH 7.17, 1H, d, J = 2.0 Hz, H-5;

9



258

3'',3''-dimethyl-2H-chromen)-6-prenyl-2H-chromen-2-one.

259

Compound 6 was isolated as a colorless powder. Its molecular formula was established as

260

C28H26O6 from the HR-ESI-MS at m/z 481.4013 [M+Na]+ (calcd. for C28H26O6Na, 481.4018),

261

indicating sixteen degrees of unsaturation. The UV spectrum showed the absorptions at λmax 220,

262

257, 286, and 353 nm, and its IR spectrum suggested the presence of hydroxyl (3346 cm-1),

263

carbonyl (1681 cm-1), aromatic ring (1628 cm-1), and methyl (1384 cm-1) functionalities. It was

264

confirmed the existence of the 3-arylcoumarin skeleton in compound 6.10

265

The 1H NMR signals (Table 2) of compound 6 including four doublets of cis-olefinic protons

266

(δH 6.63, 1H, d, J = 10.0 Hz, H-11; δH 5.69, 1H, d, J = 10.0 Hz, H-12; δH 6.61, 1H, d, J = 10.0 Hz,

267

H-1′′; δH 5.68, 1H, d, J = 10.0 Hz, H-2′′) and two singlets of dimethyl protons (δH 1.48, 6H, s,

268

14/15-CH3; δH 1.46, 6H, s, 4′′/5′′-CH3), which were in agreement with typical signals for two

269

typical dimethylchromene rings.12 Moreover, a typical 2-propenyl (δH 3.32, 2H, d, J = 6.9 Hz, H-7′;

270

δH 5.95, 1H, dd, J = 17.1, 10.3 Hz, H-8′; δH 5.08, 1H, d, J = 17.1 Hz, H-9′a; δH 5.06, 1H, d, J =

271

10.3 Hz, H-9′b) (Table 2) was assigned to H-5′ according to the HMBC correlations of H-7′/C-4′;

272

H-7′/C-6′; H-9′/C-7′ (Figure 2).13 The other fragments of compound 6 were connected by the

273

HMBC correlations of H-4/C-2; H-4/C-5; H-4/C-1′; H-5/C-11; H-8/C-6; H-11/C-13; H-1′′/C-3′′

274

(Figure 2), the 2D-NOESY correlations of H-12/15-CH3; H-2′′/5′′-CH3 (Figure 2), and the 1H-1H

275

COSY correlations of H-11/H-12; H-7′/H-8′; H-8′/H-9′; H-1′′/H-2′′ (Figure 2). Consequently,

276

compound 6 was identified as 3-[4',6'-dihydroxy-5'-(2-propenyl) -3'',3''-dimethyl-2H-chromen]-

277

14,15-dimethyl-pyrano-chromen-2-one.

278

Compound 7 was obtained as a colorless powder. It displayed a molecular ion [M+Na]+ peak

279

at m/z 483.3528 (calcd. for C28H28O6Na, 483.3523) by the HR-ESI-MS, corresponding to a

280

molecular formula of C28H28O6 with fifteen degrees of unsaturation. The UV spectrum showed the

281

absorptions at λmax 222, 257, 286, and 354 nm, and its IR spectrum suggested the presence of

282

hydroxyl (3347 cm-1), carbonyl (1681 cm-1), aromatic ring (1627 cm-1), methyl (1385 cm-1)

283

functionalities. A closer comparison of the 1H and

284

compound 6 revealed that compound 7 ought to share the 3-arylcoumarin skeleton.10 The

285

differences between compound 7 and compound 6 which displayed a single peak (δH 6.82, 1H, s,

286

H-4′) and 5′-isopropanol (δH 1.37, 6H, s, 8′/9′-CH3) in compound 7. The structure of compound 7

13

C NMR spectra (Table 3) with those of

10


Page 10 of 24

Page 11 of 24


287

was further confirmed by analyses of the HMBC correlations of H-4/C-2; H-4/C-5; H-4/C-1′;

288

H-5/C-11; H-8/C-6; H-11/C-13; H-4′/C-7′; H-4′/C-1′′; H-1′′/C-3′′ (Figure 2), the 2D-NOESY

289

correlations of H-12/15-CH3; H-2′′/5′′-CH3 (Figure 2), and the 1H-1H COSY correlations of

290

H-11/H-12; H-1′′/H-2′′ (Figure 2). Therefore, compound 7 was identified as 3-(6'-dihydroxy-5'-

291

isopropanol-3'',3''-dimethyl-2H-chromen)-14,15-dimethyl-pyrano-chromen-2-one.

292

Compound 8 was isolated as a colorless powder with the molecular formula C30H30O5

293

determined by HR-ESI-MS at m/z 493.1529 [M+Na]+ (calcd. for C30H30O5Na, 493.1522)

294

corresponding to sixteen degrees of unsaturation. The UV spectrum showed characteristic

295

absorptions (λ max 221, 258, 286, 354 nm) for the 3-arylcoumarin skeleton10, and the IR spectrum

296

showed absorption bands for hydroxyl (3347 cm-1), carbonyl (1681 cm-1), aromatic ring (1628

297

cm-1), methyl (1384 cm-1) functionalities. The 1H NMR spectrum exhibited two single peaks (δH

298

6.81, 1H, s, H-4′; δH 6.94, 1H, s, H-6′) and an isopentenol [(7′E)-9′-hydroxy-9′-methyl-7′-butenyl]

299

(δH 6.85, 1H, d, J = 16.7 Hz, H-7′; δH 6.79, 1H, d, J = 16.7 Hz, H-8′; δH 1.51, 6H, s, 10′/11′-CH3)

300

in compound 8.14 It was concluded that compound 8 was an analogue of compound 7 according to

301

their 1H and 13C NMR data (Table 3). The structure of compound 8 was determined by the HMBC

302

correlations of H-4/C-2; H-4/C-5; H-4/C-1′; H-5/C-11; H-8/C-6; H-11/C-13; H-4′/C-7′; H-4′/C-1′′;

303

H-7′/C-9′; H-8′/C-5′; H-1′′/C-3′′ (Figure 2), the 2D-NOESY correlations of H-12/15-CH3;

304

H-8′/10′-CH3; H-2′′/5′′-CH3 (Figure 2), and the 1H-1H COSY correlations of H-11/H-12; H-7′/H-8′;

305

H-1′′/H-2′′ (Figure 2). Consequently, compound 8 was detemined as 3-(5'-isopentenol-3'',3''-di-

306

methyl-2H-chromen)-14,15-dimethyl-pyrano-chromen-2-one.

307

Compound 9 was obtained as a colorless powder, and its molecular formula was determined

308

as C30H30O6 by HR-ESI-MS at m/z 509.2074 [M+Na]+ (calcd. for C30H30O6Na, 509.2077),

309

corresponding to sixteen degrees of unsaturation. The UV spectrum showed absorptions at λmax

310

220, 257, 285, 353nm and the IR spectrum showed absorption peaks at 3346, 16806, 1627, 1384

311

cm-1, which are similar to those of compound 6. It can be concluded that compound 9 was an

312

analogue of compound 6. A closer comparison of the 1H and

313

those of compound 6 revealed that compound 9 contained the 3-arylcoumarin skeleton.10 The only

314

difference between compound 9 and compound 6 was the appearance of the 5′-prenyl (δH 3.33, 2H,

315

d, J = 7.2 Hz, H-7′; δH 5.25, 1H, t, J = 7.2, 1.5 Hz, H-8′; δH 1.77, 3H, s, 10′-CH3; δH 1.66, 3H, s,

13

11


C NMR spectra (Table 3) with


Page 12 of 24

316

11′-CH3) in compound 9 and the appearance of the 5′-propenyl in compound 6 (Table 3). The

317

structure of compound 9 was established on the HMBC correlations of H-4/C-2; H-4/C-5;

318

H-4/C-1′; H-5/C-11; H-8/C-6; H-11/C-13; H-7′/C-4′; H-7′/C-6′; H-7′/C-9′; H-1′′/C-3′′ (Figure 2),

319

the 2D-NOESY correlations of H-12/15-CH3; H-8′/10′-CH3; H-2′′/5′′-CH3 (Figure 2), and the

320

1

321

was identified

322

pyrano-chromen-2-one.

H-1H COSY correlations of H-11/H-12; H-7′/H-8′; H-1′′/H-2′′ (Figure 2). Therefore, compound 9 as 3-(4',6'-dihydroxy-5'-prenyl-3'',3''-dimethyl-2H-chromen)-14,15-dimethyl-

323

Additionly, the known compounds (10-21) were identified by comparison of their

324

spectroscopic data with those reported in the references. Their structures were determined as

325

xanthyletin (10),15 xanthoxyletin (11),15 clausarin (12),15 nordentatin (13),15 4-(1-methylpropyl)-5,

326

7-dihydroxy-8-(4-hydroxy-3-methylbutyryl)-6-(3-methylbut-2-enyl)chromen-2-one (14),16 5,7-di-

327

hydroxy-8-(4-hydroxy-3-methylbutyryl)-6-(3-methylbut-2-enyl)-4-phenylchromen-2-one

328

brasimarin A (16),17 muralatin D (17),14 mammea B/BB (18),17 robustic acid (19),18 marianin A

329

(20),19 asphodelin A (21).20 Moreover, the known compounds (10-21) were obtained from this

330

plant for the first time. All the compounds (1-21) are shown in Figure 3.

(15),16

331

Statistical analysis of anti-AChE and hepatoprotective activities. The anti-AchE activities

332

of compounds (1-21) were evaluated with Tacrine as the positive control. The screening results of

333

compounds (1-21) are shown in Table 4. Compared with the control group, compound 1 and

334

compound 3 exhibited significant inhibitory activities with IC50 values of 11.23, 13.32 µM and

335

compounds 5, 6, 7, 9 exhibited weak inhibitory activities with IC50 values of 68.59, 75.02, 89.69,

336

66.30 µM, separately. In contrast, the rest of the compounds showed no inhibitory activities. The

337

anti-AChE activities of selective compounds suggested that the substituent group of 6′-OH played

338

an important role in mediating the anti-AChE activities of compounds 1-21, and further

339

Structure-Activity Relationship research would be undertaken in future experiments. Meanwhile,

340

compounds (1-21) were assayed with bicyclol (hepatoprotective activity drug) as the positive

341

control for their hepatoprotective activities against D-galactosamine-induced toxicity in HL-7702

342

cells. The results of pharmacological activities are displayed in Table 5, the inhibition (%) of

343

compounds 2, 4, 12, 15, 17, 18, and 19 based on the computing formula with values of 21.7, 32.2,

344

66.2, 35.4, 59.8, 52.2, 30.3, and 41.4, respectively. In contrast, the rest of the compounds showed 12


Page 13 of 24


345

no hepatoprotective activities. The significance of unpaired observations between normal or

346

control and tested samples was determined by Student’s t-test.21 Differences were considered

347

significant at p < 0.05.22 The study of Structure-Activity Relationship of the hepatoprotective

348

compounds needs further research.

349

■ AUTHOR INFORMATION

350

Corresponding Author

351

E-mail: [email protected]

352

Funding

353

This work was financially supported by the Key Scientific Research Project of Colleges and

354

Universities in Henan Province (Based PC12 cell — studies on neuroprotective components of

355

Magnolia biondii Pamp.), the National Natural Science Foundation of China (No.81673613), the

356

National Natural Science Foundation of China (No.81781260288), and the Science and

357

Technology Research Project of Jiangxi Provincial Education Department (No.1508ZZ).

358

Notes

359

The authors declare that there are no conflicts of interest.

360 361 362 363 364 365 366 367 368 369 370

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371

Su, Y. L. Hepatoprotective sesquiterpenes and rutinosides from Murraya koenigii (L.) Spreng. J.

372

Agr. Food Chem. 2014, 62, 4145-4151.

373

(6) Peng, X. R., Wang, X., Dong, J. R., Qin, X. J., Li, Z. R., Yang, H., Zhou, L., Qiu, M. H.

374

Rare hybrid dimers with anti-acetylcholinesterase activities from a Safflower (Carthamus

375

tinctorius L.) seed oil cake. J. Agr. Food Chem. 2017, 65, 9453-9459.

376

(7) Wang, Y. G., Ma, Q. G., Tian, J., Ren, J., Wang, A. G., Ji, T. F., Yang, J. B., Su, Y. L.

377

Hepatoprotective triterpenes from the gum resin of Boswellia carterii. Fitoterapia 2016, 109,

378

266-273.

379

(8) Ma, Q. G., Guo, Y. M., Luo, B. M., Liu, W. M., Wei, R. R., Yang, C. X., Ding, C. H., Xu,

380

X. F., He, M. H. Hepatoprotective phenylethanoid glycosides from Cirsium setosum. Nat. Prod.

381

Res. 2016, 30, 1824-1829.

382

(9) Liu, Y. F., Liang, D., Luo, H., Hao, Z. Y., Wang, Y., Zhang, C. L., Zhang, Q. J., Chen, R.

383

Y., Yu, D. Q. Hepatoprotective iridoid glycosides from the roots of Rehmannia glutinosa. J. Nat.

384

Prod. 2012, 75, 1625-1631.

385 386

(10) Fukai, T., Sheng, C. B., Horikoshi, T., Nomura, T. Isoprenylated flavonoids from underground parts of Glycyrrhiza glabra. Phytochemistry 1996, 43, 1119-1124.

387

(11) Nguyen, C. N., Trinh, B. T. D., Tran, T. B., Nguyen, L. T. T., Jäger, A. K., Nguyen, L. H.

388

D. Anti-diabetic xanthones from the bark of Garcinia xanthochymus. Bioorg. Med. Chem. Lett.

389

2017, 27, 3301-3304.

390

(12) Thepthong, P., Phongpaichit, S., Carroll,

A. R., Voravuthikunchai, S. P.

391

Mahabusarakam, W. Prenylated xanthones from the stem bark of Garcinia dulcis. Phytochem.

392

Lett. 2017, 21, 32-37.

393

(13) Li, C., Liu, H. X., Zhao, L. Y., Zhang, W. M., Qiu, S. X., Xiaoyun Yang, X. Y., Tan, H. B.

394

Antibacterial neolignans from the leaves of Melaleuca bracteata. Fitoterapia 2017, 120,

395

171-176.

396

(14) Lv, H. N., Wang, S., Zeng, K. W., Li, J., Guo, X. Y., Ferreira, D., Zjawiony, J. K., Tu, P.

397

F., Jiang, Y. Anti-inflammatory coumarin and benzocoumarin derivatives from Murraya alata. J.

398

Nat. Prod. 2015, 78, 279-285.

399

(15) Ribeiro, A. B., Abdelnur, P. V., Garcia, C. F., Belini, A., Severino, V. G. P., Silva, M. F. G. 14


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400

F., Fernandes, J. B., Vieira, P. C., Carvalho, S. A., Souza, A. A., Machado, M. A. Chemical

401

characterization of Citrus sinensis grafted on C. limonia and the effect of some isolated

402

compounds on the growth of Xylella fastidiosa. J. Agric. Food Chem. 2008, 56, 7815-7822.

403 404

(16) Scio, E., Ribeiro, A., Alves, T. M. A., Romanha, A. J., Shin, Y. G., Cordell, G. A., Zani, C.L. New bioactive coumarins from Kielmeyera albopunctata. J. Nat. Prod. 2003, 66, 634-637.

405

(17) Ito, C., Itoigawa, M., Mishina, Y., Filho, V. C., Enjo, F., Tokuda, H., Nishino, H.,

406

Furukawa, H. Chemical constituents of Calophyllum brasiliense. 2. Structure of three new

407

coumarins and cancer chemopreventive activity of 4-substituted coumarins. J. Nat. Prod. 2003, 66,

408

368-371.

409 410

(18) Hadden, M. K., Galam, L., Gestwicki, J. E., Matts, R. L., Blagg, B. S. J. Derrubone, an inhibitor of the Hsp90 protein folding machinery. J. Nat. Prod. 2007, 70, 2014-2018.

411

(19) Fukuda, T., Sudoh, Y., Tsuchiya, Y., Okuda, T., Fujimori, F., Igarashi, Y. Marianins A and

412

B, prenylated phenylpropanoids from Mariannaea camptospora. J. Nat. Prod. 2011, 74,

413

1327-1330.

414 415 416 417

(20) El-Seedi, H. R. Antimicrobial arylcoumarins from Asphodelus microcarpus. J. Nat. Prod. 2007, 70, 118-120. (21) Li, Y., Zhang, D. M., Li, J. B., Yu, S. S., Li, Y., Luo, Y. M. Hepatoprotective sesquiterpene glycosides from Sarcandra glabra. J. Nat. Prod. 2006, 69, 616-620.

418

(22) Lin, M. H., Liu, H. K., Huang, W. J., Huang, C. C., Wu, T. H., Hsu, F. L. Evaluation of

419

the potential hypoglycemic and beta-cell protective constituents isolated from Corni fructus to

420

tackle insulin-dependent diabetes mellitus. J. Agric. Food Chem. 2011, 59, 7743-7751.

421 422 423 424 425 426 15



Page 16 of 24

427 428 429 Cucumis bisexualis (15.5 kg) 90% EtOH crude extract (1.6 kg) solvent extraction

petroleum ether layer (98.6 g)

n-BuOH layer (305.8 g)

EtOAc layer (235.5 g) activity screening

active site (EtOAc layer) silica gel (100-200 mesh) column (n-hexane/EtOAc = 12:1 to 3:1)

A (29.6 g)

B (59.5 g) silica gel (100-200 mesh) column (petroleum ether/EtOAc = 15:1 to 4:1)

B1 (12.5 g)

B3 (9.8 g)

B2 (23.0 g)

430 431 432

4

12

13

14

15

1

3

silica gel (100-200 mesh) column (petroleum ether/EtOAc = 10:1 to 3:1)

C1 (12.5 g) C4 (10.6 g) C2 (26.3 g)

Sephadex LH-20 (95% ), prep-HPLC (220 nm, 6 mL/min)

Sephadex LH-20 (95% ), prep-HPLC (220 nm, 6 mL/min)

2

D (19.5 g)

C (72.8 g)

5

17 18

19

6

C3 (20.4 g)

the same way

7

21

16

8

9

10

11

Figure 1 The flow chart of compounds (1-21) for extraction and separation.

433 434 435 436 437 438 439 440 441 442 443 16


20

Page 17 of 24


444 445 446 447 448

449 450 451

Figure 2 Key HMBC, 2D NOESY, and 1H-1H COSY correlations of compounds 1-9.

452 453 454 455 456 457 458 459 460 17



Page 18 of 24

461 462 463 R1

H3 C O O

O

CH3

CH3 O

H3C O O

O

CH3

H3C

H3C CH3 R4

R2

R3

R1

R3 R2

R1

R2

R3

R4

1

OH

isopropyl

OH

2

OH

OH H

prenyl

H

3

6

R2

R3

OH

2-propenyl

OH OH

OH

7

H

isopropanol

H

8

H

isopentenol

H

9

OH

prenyl

OH

dimethylallyl OH

OH

H

5

ethyl

H

H

4

prenyl

H

H

R1

OH R1 CH3 O

CH3 O

O O

H3C R2

HO

O

O

R3

R1

R2

R3

10

H

H

H

11

H

H

H3C CH3

OH

OCH3

12

isoprenyl

isoprenyl

OH

14

13

isoprenyl

H

OH

15

R

R isobutyl phenyl

OH H3 C

O

O

HO

O

O

O

O

CH3

H3CO

CH3

CH3

18

17 O

H3C

O

OH

OCH3

CH3

O

O

O H3 C

16 O

CH3

HO H3CO

H3 C

CH3

CH3

O

O

HO

O

O

O OH

CH3 OCH3 OH

464 465 466

CH3

OCH3

19

20

Figure 3

O

CH3 CH3

Structures of compounds 1-21.

467 468 469 470 18


OH OH

21

Page 19 of 24


471 472 473 474 475 476 No.

Table 1 1 H NMR (400 MHz, Acetone-d6), 13C NMR (100 MHz, Acetone-d6), and key HMBC correlations of compounds 1-3. 1

2

δH

1

δC

13

HMBC( H - C)

3

δH

δC

1

13

HMBC( H- C)

δH

δC

HMBC(1H-13C)

1

-

-

-

-

-

-

-

-

-

2

-

160.8

-

-

160.9

-

-

160.9

-

3

-

124.9

-

-

124.8

-

-

124.8

-

4

7.53(s)

141.4

C-2,C-5,C-1′

7.54(s)

141.3

C-2,C-5,C-1′

7.54(s)

141.4

C-2,C-5,C-1′

5

7.12(s)

128.6

C-11

7.12(s)

128.6

C-11

7.16(d,2.0)

127.3

C-11

6

-

126.2

-

-

126.1

-

-

131.4

7

-

155.6

-

-

155.6

-

6.98(dd,8.0,2.0)

119.5

C-11

-

8

6.75(s)

106.3

C-10

6.76(s)

106.3

C-10

6.85(d,8.0)

128.0

C-10

9

-

155.8

-

-

155.7

-

-

151.6

-

10

-

115.2

-

-

115.3

-

-

117.8

-

11

3.34(d,7.2)

28.8

-

3.34(d,7.2)

28.8

-

3.35(d,7.2)

28.5

-

12

5.27(t,7.2,1.5)

123.3

C-6

5.26(t,7.2,1.5)

123.2

C-6

5.28(t,7.2,1.5)

123.1

-

13

-

131.3

-

-

131.3

-

-

131.5

-

14-CH3

1.77(s)

24.8

-

1.76(s)

24.8

-

1.77(s)

24.7

-

15-CH3

1.65(s)

18.9

-

1.65(s)

18.8

-

1.66(s)

18.8

-

1′

-

118.8

-

-

118.9

-

-

118.7

-

2′

-

153.7

-

-

152.6

-

-

152.7

-

3′

-

115.4

-

-

114.9

-

-

114.8

-

4′

-

156.4

-

6.81(s)

125.5

C-7′

6.79(s)

126.7

C-7′

5′

-

116.1

-

-

128.4

-

-

122.4

-

6′

-

156.4

-

6.94(s)

125.6

-

-

157.2

-

7′

2.68(m)

25.6

C-4′,C-6′

3.32(d,7.2)

29.1

C-9′

3.33(d,7.2)

29.8

C-9′

8′

1.14(s)

21.5

-

5.24(t,7.2,1.5)

123.4

C-5′

5.24(t,7.2,1.5)

123.9

C-5′

9′

1.14(s)

21.5

-

-

131.2

-

-

131.3

-

10′

-

-

-

1.76(s)

24.9

-

1.76(s)

24.9

-

11′

-

-

-

1.66(s)

18.8

-

1.66(s)

18.8

-

1′′

6.60(d,10.0)

115.8

C-4′,C-3′′

6.62(d,10.0)

115.7

C-4′,C-3′′

6.61(d,10.0)

115.8

C-4′,C-3′′

2′′

5.68(d,10.0)

128.2

-

5.67(d,10.0)

128.2

-

5.67(d,10.0)

128.3

-

3′′

-

79.3

-

-

79.2

-

-

79.3

-

4′′

1.46(s)

28.5

-

1.45(s)

28.6

-

1.46(s)

28.5

-

5′′

1.46(s)

28.5

-

1.45(s)

28.6

-

1.46(s)

28.5

-

477 478 479 480 481 19



482 483 484 485 486 487 No.

Page 20 of 24


5

δH

δC

1

-

-

2

-

3 4 5

1

13

HMBC( H - C)

6 1

13

δH

δC HMBC(1H-13C)

δH

δC

HMBC( H- C)

-

-

-

-

-

160.9

-

-

160.8

-

-

-

124.9

-

-

124.9

-

-

124.9

-

7.54(s)

141.4

C-2,C-5,C-1′

7.53(s)

141.4

C-2,C-5,C-1′

7.53(s)

141.3

C-2,C-5,C-1′

7.17(d,2.0)

127.5

C-11

7.17(d,2.0)

127.4

C-11

7.13(s)

120.6

C-11

-

-

160.8

-

6

-

131.3

-

-

131.4

-

-

120.1

-

7

6.98(dd,8.0,2.0)

119.2

C-11

6.98(dd,8.0,2.0)

119.4

C-11

-

153.3

-

8

6.86(d,8.0)

128.1

C-10

6.87(d,8.0)

128.0

C-10

6.77(s)

107.7

C-6

9

-

151.2

-

-

154.1

-

-

155.5

-

10

-

117.6

-

-

117.7

-

-

115.4

-

11

3.35(d,7.2)

28.6

-

3.34(d,7.2)

28.5

-

6.63(d,10.0)

12

5.27(t,7.2,1.5)

123.2

-

5.27(d,7.2,1.5)

123.1

-

5.69(d,10.0)

13

-

131.4

-

-

131.5

-

14-CH3

1.77(s)

24.7

-

1.76(s)

24.8

-

121.2

C-13

127.4

-

79.5

-

1.48(s)

28.4

-

1.48(s)

-

15-CH3

1.66(s)

18.9

-

1.66(s)

18.9

-

28.4

-

1′

-

118.8

-

-

118.7

-

-

118.8

-

2′

-

152.6

-

-

153.6

-

-

153.7

-

3′

-

114.9

-

-

115.5

-

-

115.6

-

4′

6.82(s)

125.6

C-7′

-

156.5

-

-

156.5

-

5′

-

128.5

-

-

122.5

-

-

109.3

-

6′

6.93(s)

125.6

C-7′

-

156.5

-

-

156.5

-

7′

2.68(m)

29.4

-

-

40.8

-

3.32(d,6.9)

39.3

C-4′,C-6′

8′

1.19(s)

14.7

-

6.31(dd,17.5,10.7)

148.1

-

5.95(dd,17.1,10.3)

138.3

-

9′a

-

-

5.02(dd,17.5,1.3)

110.8

C-7

5.08(d,17.1)

115.7

C-7′

9′b

-

-

-

5.00(dd,10.7,1.3)

110.8

C-7

5.06(d,10.3)

115.7

C-7′

10′

-

-

-

1.53(s)

26.6

-

11′

-

-

-

1.53(s)

26.6

-

1′′

6.62(d,10.0)

115.7

C-4′,C-3′′

6.60(d,10.0)

115.7

C-4′,C-3′′

6.61(d,10.0)

2′′

5.67(d,10.0)

128.2

-

5.68(d,10.0)

128.3

-

5.68(d,10.0)

3′′

-

79.3

-

-

79.3

-

79.3

-

4′′

1.46(s)

28.6

-

1.45(s)

28.5

-

1.46(s)

28.6

-

5′′

1.46(s)

28.6

-

1.45(s)

28.5

-

1.46(s)

28.6

-

488 489 490 491 20


-

-

-

-

24.9 115.8

C-3′′

128.3

-

Page 21 of 24


492 493 494 495 496 497 No.


8

δH

1

δC

13

HMBC( H - C)

9

δH

δC

1

13

HMBC( H- C)

δH

δC -

HMBC(1H-13C)

1

-

-

-

-

-

-

-

2

-

160.9

-

-

160.8

-

-

3

-

124.8

-

-

124.9

-

-

124.8

-

4

7.54(s)

141.3

C-2,C-5,C-1′

7.53(s)

141.4

C-2,C-5,C-1′

7.54(s)

141.3

C-2,C-5,C-1′

5

7.12(s)

120.7

C-11

7.12(s)

120.6

C-11

7.13(s)

120.7

C-11

160.9

-

-

6

-

120.1

-

-

120.2

-

120.1

-

7

-

153.2

-

-

153.3

-

-

153.2

-

8

6.76(s)

107.6

C-6

6.77(s)

107.7

C-6

6.76(s)

107.6

C-6

9

-

155.4

-

-

155.5

-

-

155.3

-

10

-

115.4

-

-

115.3

-

-

115.4

-

11

6.64(d,10.0)

121.2

C-13

6.63(d,10.0)

121.1

C-13

6.64(d,10.0)

121.3

C-13

12

5.69(d,10.0)

127.5

-

5.68(d,10.0)

127.4

-

5.69(d,10.0)

127.5

-

13

-

79.4

-

-

79.5

-

-

79.4

-

14-CH3

1.48(s)

28.4

-

1.47(s)

28.5

-

1.48(s)

28.4

-

15-CH3

1.48(s)

28.4

-

1.47(s)

28.5

-

1.48(s)

28.4

-

1′

-

118.7

-

-

118.9

-

-

118.8

-

2′

-

153.7

-

-

153.6

-

-

153.7

-

3′

-

115.8

-

-

115.7

-

-

115.6

-

4′

6.82(s)

126.7

C-7′,C-1′′

6.81(s)

126.0

C-7′,C-1′′

-

156.5

-

5′

-

122.4

-

-

128.6

-

-

110.1

-

6′

-

157.3

-

6.94(s)

125.4

-

-

156.5

7′

-

77.3

-

6.85(d,16.7)

119.8

C-9′

3.33(d,7.2)

29.5

8′

1.37(s)

23.1

-

6.79(d,16.7)

138.2

C-5′

5.25(t,7.2,1.5)

123.6

9′

1.37(s)

23.1

-

-

82.6

-

131.3

-

10′

-

-

-

1.51(s)

24.6

-

1.77(s)

24.9

-

11′

-

-

-

1.51(s)

24.6

-

1.66(s)

18.8

-

1′′

6.61(d,10.0)

115.7

C-3′′

6.62(d,10.0)

115.8

C-3′′

6.61(d,10.0)

115.7

2′′

5.67(d,10.0)

128.2

-

5.67(d,10.0)

128.3

-

5.68(d,10.0)

128.2

-

3′′

-

79.2

-

-

79.3

-

-

79.2

-

4′′

1.46(s)

28.5

-

1.46(s)

28.6

-

1.45(s)

28.5

-

5′′

1.46(s)

28.5

-

1.46(s)

28.6

-

1.45(s)

28.5

-

498 499 500 501 502 21


-

C-4′,C-6′,C-9′ -

C-3′′


503 504 505 506

507

Table 4 Anti-AChE activities of selective compounds. Compound TAa 1

Page 22 of 24

IC50 (µM) 0.35 11.23

3

13.32

5

68.59

6

75.02

7

89.69

9

66.30

a

Tacrine, positive control for anti-AChE activity.

508 509 510 511 512

Table 5 Hepatoprotective effects of selective compounds (10µM). compound normal control bicyclol 2 4 12 15 17 18 19

513 514 515

cell survival rate (% of normal)

Inhibition (% of control)

100 .0 ± 6.3 42.3 ± 2.6 54.8 ± 7.6* 60.9 ± 3.9* 80.5 ± 5.7* 62.7 ± 7.3* 76.8 ± 1.6* 72.4 ± 2.8* 59.8 ± 6.4* 66.2 ± 4.7*

21.7 32.2 66.2 35.4 59.8 52.2 30.3 41.4

Results were expressed as means ± SD (n= 3; for normal and control, n = 6); bicyclol was used as positive control (10µM). *p< 0.05.

516 517 518 519 22


Page 23 of 24


520 521 522

The TOC Graphic

523 524 525

23



152x69mm (96 x 96 DPI)


Page 24 of 24

Molecular Characterization and Bioactivity of Coumarin Derivatives

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