Coumarin Analogues from the Citrus grandis (L.) Osbeck and Their

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Bioactive Constituents, Metabolites, and Functions

Coumarin Analogues from the Citrus grandis (L.) Osbeck and Their Hepatoprotective Activity Danmei Tian, Fangfang Wang, Menglong Duan, Lingyun Cao, Youwei Zhang, Xin-Sheng Yao, and Jinshan Tang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06489 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 30, 2019

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Page 1 of 38

Journal of Agricultural and Food Chemistry

Coumarin Analogues from the Citrus grandis (L.) Osbeck and Their Hepatoprotective Activity

Danmei Tian, †,‡,‖ Fangfang Wang, †,‡,‖ Menglong Duan,†,§ Lingyun Cao,†,§ Youwei Zhang,# Xinsheng Yao,†,‡,* and Jinshan Tang †,‡,*

†

Institute of Traditional Chinese Medicine and Natural Products, College of

Pharmacy, Jinan University, Guangzhou 510632, People’s Republic of China ‡

Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM

and New Drug Research, Jinan University, Guangzhou 510632, People’s Republic of China §

Key Laboratory of Standard Material in Natural Medicine of Guangdong Province,

Guangzhou Xiangxue Pharmaceutical Ltd. Co., Guangzhou 510663, China #

Department of Pharmacology, Case Comprehensive Cancer Center, Case Western

Reserve University School of Medicine, Cleveland, OH 44106, USA

1

ACS Paragon Plus Environment


Page 2 of 38

1

ABSTRACT:

2

Seven new coumarin analogues (1, 2, 4-8), together with ten known analogues (3,

3

9-17), were isolated from the air-dried pericarp of Citrus grandis. The structures of

4

these compounds were determined by HR-ESI-MS, UV/vis, and 1D- and 2D-NMR

5

spectra. Meanwhile, the hepatoprotective activities of all these coumarins were

6

evaluated by MTT assays using the D-galactosamine-induced LO2 cell injury model.

7

The results show that compounds 3 and 4 exhibited the strongest hepatoprotective

8

activities. Moreover, compounds 3 and 4 suppressed the increases in the levels of

9

alanine

transaminase

(ALT)

and

aspartate

transaminase

(AST)

in

10

D-galactosamine-treated

LO2 cells, further confirming the hepatoprotective effects of

11

these compounds. Mechanistically, compounds 3 and 4 increased the activities of

12

antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase

13

(GSH-Px), and decreased the level of malondialdehyde (MDA) in injured LO2 cells

14

induced by D-galactosamine. These findings shed light on a better understanding of

15

the hepatoprotective effect of Citrus grandis, providing novel insights into the

16

development of coumarin-based hepatoprotective drugs in the future.

17 18

KEYWORDS:

19

Citrus grandis (L.) Osbeck, Coumarin, Hepatoprotective, Alanine transaminase

20

(ALT), Aspartate transaminase (AST), Antioxidant enzyme

21 22 2


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23

■ INTRODUCTION

24

Citrus grandis (L.) Osbeck, commonly known as pomelo, ahaddock or limau

25

bali, is an important cultivated Citrus species belonging to the Rutaceae family, which

26

is native to southeast Asia and China.1 The dried pericarps of immature or

27

near-mature Citrus grandis （ L. ） Osbeck together with its cultivar Citrus grandis

28

‘Tomentosa’ are recorded in the Chinese Pharmacopoeia (ChP) as “Citri Grandis

29

Exocarpium” (huajuhong), and have been used as a Traditional Chinese Medicine for

30

the treatment of cough with asthma, abdominal pain and stomach ache for centuries.2

31

In addition, Citrus grandis is also an important human food source, whose fresh fruits

32

and derived products have been used as essential ingredients in human diet.3-5

33

Chemical investigations of Citrus grandis revealed components including flavonoids6,

34

coumarins6-9, limonins10, terpenoids11, alkaloids6,8,9,12, and so on. Coumarins have

35

been shown to possess a wide range of biological activities including anti-

36

inflammatory13,

37

antimicrobial6, and hepatoprotection15-17. Despite the fact that coumarin is one of the

38

mostly rich constituents from the plant of Citrus grandis,11,18 little attention has been

39

paid to the hepatoprotective activities of coumarins isolated from Citrus grandis（L.)

40

Osbeck.

neuroprotective13,

antiproliferation14,

glucose

consumption14,

41 42

In this study, we isolated seven new coumarins (1, 2, 4-8), together with ten

43

known analogues (3, 9-17), from the air-dried pericarp of Citrus grandis, which

44

include two rare coumarins (1 and 2) that contain 3-hydroxy-3-methylglutaric acid 3



45

(HMG) group in the sugar moiety. The structures of all the compounds were

46

identified by extensive UV, IR, MS, NMR spectroscopic data. Further, the

47

hepatoprotective effects of all these compounds were evaluated by the

48

D-galactosamine

49

of the cells with coumarins (20 μM) significantly alleviated the cell survival inhibition

50

induced by

51

survival inhibition induced by

52

compounds 3 and 4 suppressed the increase in ALT and AST levels in

53

D-galactosamine-treated

54

compounds. We further investigated the mechanisms by which these coumarins

55

protect liver cell injury caused by D-galactosamine and reveal that they modulate the

56

enzymatic activities of antioxidant enzymes that clears reactive oxygen species and

57

oxidative lipid products.

-induced LO2 cell injury model. Our studies show that pretreatment

D-galactosamine.

Two potent compounds 3 and 4 inhibited the cell D-galactosamine

in a range of doses. Further,

LO2 cells, illustrating hepatoprotective effects of these

58 59

■ MATERIALS AND METHODS

60

General Experimental Procedures. 1D- and 2D-NMR spectra were performed

61

with a Bruker AV 600 (Bruker Co. Ltd., Bremen, German) using solvent signals

62

(DMSO-d6: H 2.50 / δC 39.5; CD3OD: H 3.31 / δC 49.0) as internal reference. IR

63

spectra were obtained on a JASCO FT/IR-480 plus spectrometer (JASCO

64

International Co. Ltd., Tokyo, Japan). UV/vis spectra were acquired on a JASCO

65

V-550 UV/Vis spectrometer (JASCO International Co. Ltd., Hachioji, Tokyo, Japan)

66

and HR-ESI-MS spectra were obtained on a Waters Synapt G2 mass spectrometer 4


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Page 5 of 38


67

(Waters, Manchester, U.K.). HPLC analyses were performed on a Waters 2695

68

separation module (Waters, Manchester, U.K.) equipped with a 2998 photodiode

69

array detector (Waters, Manchester, U.K.) and an Alltech 3300 evaporative light

70

scattering detector (Alltech Inc., Deerfield, Illinois, U.S.) using a Phenomenex

71

Gemini C18 column (5 μm, ϕ 4.6 × 250 mm; Phenomenex Inc., Torrance, Calif, U.S.).

72

The semi-preparative and preparative HPLC were carried out on a Waters 1515

73

isocratic HPLC pump (Waters, Manchester, U.K.) coupled to a 2489 UV/vis detector

74

(Waters, Manchester, U.K.) using a Phenomenex Gemini C18 column (5 μm, ϕ 10 ×

75

250 mm; Phenomenex Inc., Torrance, Calif, U.S.).

76

CH3OH for HPLC was purchased from BCR International Co. Ltd. (Shanghai,

77

China). Acetonitrile (CH3CN) was purchased from Merck (Darmstadt, Germany).

78

Reference substances for sugar analysis were purchased from Sigma Aldrich

79

(Shanghai,

80

tripyrrolidinophosphonium hexafluorophosphate (PyBOP), 1-hydroxybenzotriazole

81

(HOBt), N, N-dimethylformamide (DMF), LiBH4, acetic acid and tetrahydrofuran

82

(THF) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China).

83

Triethylamine (Et3N) was purchased from Tianjin Ke-miou Chemical Reagent Co.,

84

Ltd. (Tianjin, China). Bicyclol was purchased from Target Molecule Corp. (Target

85

Mol, Boston, U.S.).

86

Technology Co., Ltd. (Shanghai, China). Silica gel (200-300 mesh, Qingdao Marine

87

Chemical Ltd., Shandong, China), macroporous absorption resin HP20 (Mitsubishi

88

Chemical Co., Tokyo, Japan), octadecylsilanized (ODS) (12 nm, 50 μm, YMC Ltd.,

China).

β-Phenylethylamine,

D-Galactosamine

(benzotriazol-1-yloxy)

was purchased from Meryer Chemical

5



89

Tokyo, Japan) and Sephadex LH-20 (Amersham Pharmacia Biotech, Sweden) were

90

used for column chromatography (CC). TLC was performed on pre-coated silica gel

91

plates (SGF254, 0.2 mm, Yantai Chemical Industry Research Institute, Shandong,

92

China).

93

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS)

94

were purchased from Gibco (New York, USA). DMSO and methyl thiazolyl

95

tetrazolium (MTT) were purchased from Aladdin Reagent Co., Ltd. (Shanghai,

96

China). The LO2 cell line was provided by Professor Dongmei Zhang, College of

97

Pharmacy, Jinan University. Detection kits for malondialdehyde (MDA), superoxide

98

dismutase (SOD), and glutathione peroxidase (GSH-Px) were purchased from

99

Beyotime Institute of Biotechnology (Shanghai, China). The ALT and AST activities

100

were measured with HITACHI automatic analyzer 7600.

101

Plant Material. The dried fruits of Citrus grandis (L.) Osbeck were provided by

102

Xiangxue Pharmaceutical Co., Ltd. R&D Center ， Guangdong province, China, in

103

September 2016 and authenticated by Mr. Minmei Chen, the manager of Quality

104

Control Department, Xiangxue Pharmaceutical Co., Ltd. A voucher specimen (no.

105

JNU-CIJR-2016) was deposited in Xiangxue Pharmaceutical Co., Ltd. Academician

106

workstation.

107

Extraction and Isolation. Air-dried shredded pieces of Citrus grandis (L.)

108

Osbeck (19.0 kg) were refluxed twice with 70% EtOH-H2O (160.0 L, 1.5 h each time)

109

to get crude extract (5.6 kg, yield 29.5%). The crude extract (5.2 kg) was suspended in

110

H2O (10.0 L) and subjected to column chromatography over a HP-20 macroporous 6


Page 6 of 38

Page 7 of 38


111

resin (ϕ 20.0 × 85.0 cm) eluted with EtOH-H2O (0, 30, 50 and 95%) to afford four

112

fractions CIGR-A~CIGR-D.

113

The 50% (v/v) EtOH-H2O elution portion CIGR-C (483 g) was subjected to silica

114

gel column chromatography eluted with CHCl3-CH3OH-H2O (100:0:0-80:20:2, v/v/v)

115

to afford 16 subfractions CIGR-C-1~CIGR-C-16. The subfraction CIGR-C-2 (39.2 g)

116

was further separated by ODS column chromatography (CH3OH-H2O, 35:65 to 45:55,

117

v/v) to obtain 5 subfractions (CIGR-C-2-A~CIGR-C-2-E). Compound 10 (12.5 g) was

118

recrystallized from subfraction CIGR-C-2-A (25.6 g) and the subfraction

119

CIGR-C-2-D (2.7 g) was subjected to preparative HPLC with 24% CH3CN-H2O

120

(0.1% acetic acid) elution to yield compound 12 (21.3 mg, tR = 14.0 min). The

121

subfraction CIGR-C-3 (2.1 g) was isolated by Sephadex LH-20 column

122

chromatography eluted with 80% CH3OH-H2O and subsequently applied to

123

preparative HPLC with 24% CH3CN-H2O (0.1% acetic acid) elution to yield

124

compound 5 (17.7 mg, tR = 11.0 min). The subfraction CIGR-C-4 (3.9 g) was

125

separated by ODS column chromatography eluted with CH3OH-H2O (30:70 to 45:55,

126

v/v)

127

CIGR-C-4-B (0.3 g) was applied to preparative HPLC eluted with 24% CH3CN-H2O

128

(0.1% acetic acid) to yield 15 (1.8 mg, tR = 19.2 min). The subfraction CIGR-C-4-E

129

(0.4 g) was subjected to preparative HPLC eluted with 24% CH3CN-H2O (0.1% acetic

130

acid) to afford 4 (88.5 mg, tR = 8.6 min). The subfraction CIGR-C-4-F (0.3 g) was

131

purified by preparative HPLC with 24% CH3CN-H2O (0.1% acetic acid) as the eluent

132

to obtain compounds 6 (5.8 mg, tR = 16.2 min), 8 (1.3 mg, tR = 10.8 min), 16 (205.4

to

yield

12

subfractions

CIGR-C-4-A~CIGR-C-4-L.

7


The

subfraction


Page 8 of 38

133

mg, tR = 20.8 min), and 17 (34.1 mg, tR = 22.2 min). The subfraction CIGR-C-4-H

134

(0.4 g) was employed to preparative HPLC with 24% CH3CN-H2O (0.1% acetic acid)

135

as the eluent to afford 9 (26.0 mg, tR = 16.6 min) and 11 (246.7 mg, tR = 24.0 min).

136

The subfraction CIGR-C-5 (7.6 g) was separated with ODS column chromatography

137

(CH3OH-H2O, 20: 80 to 40: 60, v/v) and subsequently purified by preparative HPLC

138

with 24% CH3CN-H2O (0.1% acetic acid) as the eluent to obtain compounds 3 (12.2

139

mg, tR = 7.8 min), 7 (16.3 mg, tR = 17.6 min), and 14 (24.3 mg, tR = 9.5 min). The

140

subfraction CIGR-C-7 (5.2 g) was isolated with ODS column chromatography

141

(CH3OH-H2O,

142

(CIGR-C-7-A~CIGR-C-7-F). The subfraction CIGR-C-7-B (1.7 g) was isolated by

143

Sephadex LH-20 (80% CH3OH-H2O) to obtain CIGR-C-7-B-1~CIGR-C-7-B-7. The

144

subfraction CIGR-C-7-B-3 (1.4 g) was subjected to semipreparative HPLC eluted

145

with 24% CH3CN-H2O (0.1% acetic acid) to obtain 2 (4.8 mg, tR = 12.2 min) and 13

146

(111.9 mg, tR = 7.3 min). The subfraction CIGR-C-7-C (1.0 g) was subjected to

147

semipreparative HPLC eluted with 24% CH3CN-H2O (0.1% acetic acid) to afford 1

148

(180.9 mg, tR = 13.4 min).

20:

80

to

100:

0,

v/v)

to

obtain

6

sufractions

27

149

Columbianoside Ⅰ (1). Yellow amorphous solid; [α] D -80.4 (c 0.5, CH3OH);

150

UV (CH3OH) max (log ): 206 (4.6), 223 (4.2), 261 (3.8), 326 (4.2); IR (KBr) max:

151

3430, 1723, 1622, 1256 cm-1; HR-ESI-MS: m/z 575.1743 [M + Na]+ (calcd for

152

C26H32O13Na, 575.1741); 1H- and 13C-NMR spectral data (Table 1).

153 154

29

Columbianoside Ⅱ (2). Yellow amorphous solid; [α] D -114.2 (c 0.5, CH3OH); UV (CH3OH) max (log ): 208 (4.6), 261 (3.7), 327 (4.2); IR (KBr) max: 3412, 1720, 8


Page 9 of 38


155

1391, 1256, 1077 cm-1; HR-ESI-MS m/z: 553.1928 [M + H]+ (calcd for C26H33O13,

156

553.1921); 1H- and 13C-NMR spectral data (Table 1). 29

157

Meranzin hydrate Ⅰ (4). Yellow amorphous solid; [α] D -40.5 (c 0.5, CH3OH);

158

UV (CH3OH) max (log ): 208 (4.6), 262 (4.1), 324 (4.2); IR (KBr) max: 3383, 2979,

159

1693, 1610, 1456, 1141 cm-1; HR-ESI-MS m/z: 317.1004 [M + Na]+ (calcd for

160

C15H18O6Na, 317.1001); 1H- and 13C-NMR spectral data (Table 1). 29

161

Meranzin hydrate Ⅱ (5). Yellow colloidal solid; [] D -22.2 (c 0.5, CH3OH); UV

162

(CH3OH) max (log ): 207 (4.5), 322 (4.1); IR (KBr) max: 3424, 1703, 1265 cm-1;

163

HR-ESI-MS m/z: 317.1002 [M + Na]+ (calcd for C15H18O6Na, 317.1001); 1H- and

164

13C-NMR

spectral data (Table 2). 29

165

Meranzin hydrate Ⅲ (6). Yellow amorphous solid; [α] D -8.4 (c 0.5, CH3OH);

166


167

1607, 1254, 1094, 1031 cm-1; HR-ESI-MS m/z: 505.1692 [M + Na]+ (calcd for

168

C23H30O11Na, 505.1686); 1H- and 13C-NMR spectral data (Table 2). 29

169

Paniculin III (7). Yellow amorphous solid; [] D -15.8 (c 0.5, CH3OH); UV

170

(CH3OH) max (log ): 206 (4.6), 221 (4.0), 259 (3.6), 323 (4.1); IR (KBr) max: 3401,

171

1717, 1602, 1036 cm-1; HR-ESI-MS m/z: 447.1631 [M + Na]+ (calcd for C21H28O9Na,

172


173

Meranzin hydrate Ⅳ (8). Yellow amorphous solid; [α] D 70.0 (c 0.5, CH3OH);

174


175

1388, 1256, 1100 cm-1; HR-ESI-MS m/z: 433.1482 [M + Na]+ (calcd for C20H26O9Na,

176




Page 10 of 38

177

Acid Hydrolysis of Compounds 1, 2, and 6-8 and Determination of the

178

Absolute Configuration of Resulting Sugars. The absolute configurations of the

179

sugar units in 1, 2, and 6-8 were identified via the method developed by Tanaka et

180

al.19 Compounds 1, 2, and 6-8 (each 1-2 mg) were hydrolyzed using 2 M HCl (2 mL)

181

for 2 h at 90 °C. The reaction mixtures were extracted with EtOAc (2 mL  2). The

182

aqueous phases were concentrated, and L-cysteine methyl ester hydrochloride (2.5

183

mg) in pyridine was added. The reaction mixtures were maintained at 60 °C and

184

reacted for 1 h. Then, a 5 μL solution of o-tolyl isothiocyanate was added to the

185

reaction mixture and heated at 60 °C for 1 h. The reaction mixture was directly

186

analyzed by HPLC. Analytical HPLC was acquired on a Phenomenex Gemini C18

187

column (5 μm, ϕ 4.6 × 250 mm) at 35 °C with a 25% CH3CN-H2O (0.01% formic

188

acid) solvent system for 32 min (0.8 mL/min). The reference sugars,

189

L-glucose, D-arabinose

190

procedure mentioned above, and all the derivatives were monitored with a UV

191

detector at 250 nm. The derivatives of D-glucose in 1, 2, 6 and 7 and L-arabinose in 8

192

were identified by comparison of the retention times with those of the reference

193

sugars. The reference sugar derivatives were recorded at 17.0 (L-glucose), 18.4

194

(D-glucose), 22.6 (L-arabinose), and 24.2 (D-arabinose) min.

195

D-glucose,

and L-arabinose, were employed to the same derivatization

Determination of the Absolute Configuration of HMG in Compound 1.

196

β-Phenylethylamine

(156.0

μmol),

triethylamine

197

(benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP, 83.8

198

μmol), and 1-hydroxybenzotriazole (HOBt, 116.8 μmol) were added to a solution of 10


(Et3N,

213.4

μmol),

Page 11 of 38


199

compound 1 (57.0 μmol) in 0.6 mL of DMF in ice bath. The mixture was stirred to

200

react for 9 h at room temperature and then quenched with diluted aqueous HCl. A

201

yellowish residue was yielded after drying under N2 gas. The residue was subjected to

202

silica gel column chromatography (CHCl3/CH3OH, 100:0, 97:3, 95:5, 90:10, 85:15,

203

70:30 and 0:100) to obtain amide A (50.3 μmol). Then, LiBH4 (900.0 μmol) was

204

added to the solution of amide A (50.3 μmol) in THF (0.5 mL) in ice bath. The

205

solution was stirred for 24 h at room temperature, and then the reaction was quenched

206

with dilute aqueous HCl. The resultant mixture was extracted with EtOAc and

207

subjected to silica gel column chromatography (CHCl3/CH3OH, 100:0, 97:3, 95:5,

208

90:10, 80:10 and 0:100) to obtain six fractions (B-1~B-6) and the fraction B-5 was

209

purified by preparative HPLC eluted with 24% CH3CN-H2O to afford B (33.5 μmol)

210

as a colorless oil (Scheme 1), which was identified as 1-β-phenylethyl-mevalonamide

211

by 1H- and

212

confirmed by comparison the optical rotation {[α] D -6.2 (c 0.5, EtOH)} and 1H- and

213

13C-NMR

13C-NMR

data.20 The R configuration of compound B was finally 27

data with that of (R)-(-)-mevalonolactone.21

214

(3R)-1-β-Phenylethyl-mevalonamide (B) Colorless oil; HR-ESI-MS m/z:

215

252.1595 [M + H]+ (calcd for C14H22NO3, 252.1600); 1H-NMR data (CD3OD, 600

216

MHz): δH 7.19-7.30 (5H, m, C6H5-2′), 3.73 (2H, m, H-5), 3.44 (2H, t, J = 7.2 Hz,

217

H-1′), 2.82 (2H, t, J = 7.2 Hz, H-2′), 2.34, 2.37 (each 1H, d, J = 14.4 Hz, H-2), 1.75

218

(2H, m, H-4), 1.21 (3H, s, H-6). 13C-NMR (CD3OD, 150 MHz): δC 174.0 (C-1), 140.4

219

(C-1′′), 129.8 (×2, C-3′′ and 5′′), 129.5 (×2, C-2′′ and 6′′), 127.4 (C-3′′), 72.1 (C-3),

220

59.2 (C-5), 47.9 (C-2), 44.3 (C-4), 41.8 (C-1′), 36.5 (C-2′), 27.5 (-CH3). 11



221

LO2 Cell Culture. Normal human hepatic LO2 cells were cultured in

222

Dulbecco’s modified eagle medium (DMEM) containing fetal bovine serum (FBS,

223

10%), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C under a 5% CO2

224

atmosphere. The cells in the logarithmic phase were used for the experiment.

225

Hepatoprotective Assay. The isolated compounds were analyzed and quantified

226

by HPLC before use, and all the compounds have a ≥ 95% purity based on the peak

227

area analysis (see Supporting Information). The compounds 1-17 were assessed for

228

their hepatoprotective activities against D-galactosamine-induced toxicity in LO2 cells

229

by an MTT method. Cell suspension with 5 × 103 cells in 100 μL of complete DMEM

230

was placed in a 96-well microplate and precultured for 24 h at 37 °C under a 5% CO2

231

atmosphere. The culture media were replaced with fresh ones (100 μL) containing

232

bicyclol or test compounds and incubated for 4 h. The cells were then exposed to 40

233

mM

234

media were aspirated and replaced with compound-free 100 μL DMEM full media

235

containing 0.5 mg/mL MTT and incubated for another 4 h. The resulting formazan

236

was dissolved in 150 μL of DMSO after aspiration of the culture medium. The optical

237

density (OD) of the formazan solution was measured on a microplate reader at 492

238

nm, and the growth inhibition (% of model) was calculated as inhibition (%) = [(OD

239

(sample) – OD (model)) / (OD (normal) – OD (model))] × 100.16,22

D-galactosamine

for 24 h. To detect cell viability, the compound-containing

240

Morphological observation. 5 × 105 LO2 cells was placed in a 6-well

241

microplate and precultured for 24 h at 37 °C under 5% CO2 atmosphere. Fresh

242

medium (1.5 mL) containing bicyclol, 3 or 4 (20 μM) was added, and the cells were 12


Page 12 of 38

Page 13 of 38


243

further cultured for another 4 h. Then, the cultured cells were exposed to 40 mM

244

D-galactosamine

245

recorded by Inverted Microscope.

for 24 h and the morphology of LO2 cells was visualized and

246

Assays for measuring ALT and AST activities. The LO2 cells were treated as

247

described above, the cell supernatants were collected to measure the ALT and AST

248

activities with HITACHI automatic analyzer 7600. The activities of ALT and AST

249

were expressed as U/L.

250

Assay for SOD and GSH-Px activities and the content of MDA. The LO2

251

cells were seeded in a 6 cm culture dish at the concentration of 3 × 106 cells per dish

252

and precultured for 24 h. Fresh medium (3 mL) containing 3 or 4 (20 μM) was added,

253

and the cells were cultured for 4 h. Then, the cultured cells were exposed to 40 mM

254

D-galactosamine

255

pooled in PBS solution and homogenized. The homogenate was centrifuged for 10

256

min at 12000 × g at 4 C. The protein contents of supernatants were measured using a

257

bovine serum albumin (BCA) protein measurement kit according to the

258

manufacturer’s instructions. The activities of SOD and GSH-Px and the content of

259

MDA in the supernatant were measured using an assay kit (Beyotime Institute of

260

Biotechnology) according to the manufacturer’s protocols.

for 24 h. After that, the cultures were washed with ice cold PBS,

261

Statistical analysis. All data were expressed as mean ± mean squared error

262

(S.M.E.) from at least three independent experiments and analyzed by one-way

263

ANOVA using GraphPad Prism (version 5.0). p < 0.05 was considered statistically

13



264

Page 14 of 38

significant.

265 266

■ RESULTS AND DISCUSSION

267

Structural Elucidation of New Compounds. The procedure for the isolation of

268

compounds began with refluxing the air-dried shredded pieces of Citrus grandis with

269

70% EtOH-H2O. The resulting extracts were employed to various column

270

chromatographic separation including macroporous absorption resin HP-20, silica gel,

271

ODS, Sephadex LH-20 column chromatography, and preparative RP HPLC, leading

272

to the purification of compounds 1-17 (Figure 1).

273

Compound 1 was isolated as yellow amorphous solid. The HR-ESI-MS showed a

274

quasimolecular ion at m/z 575.1743 [M + Na]+, indicating a molecular formula of

275

C26H32O13 and accounting for eleven degrees of unsaturation. Compound 1 showed

276

the UV absorption bands at 206, 223, 261, 326 nm and IR absorption at 3430 cm-

277

(hydroxy group) and at 1723 cm- (carbonyl group), suggesting that 1 is a

278

7-oxygenated coumarin.23 The 1H-NMR spectrum of 1 displayed two pairs of doublets

279

at H 6.23 (1H, d, J = 9.5 Hz, H-3) and H 7.96 (1H, d, J = 9.5 Hz, H-4), and at H 6.82

280

(1H, d, J = 8.6 Hz, H-6) and H 7.48 (1H, d, J = 8.6 Hz, H-5), characteristic of a 7,

281

8-disubstituted coumarin. In addition, germinal methylene proton signals at H 3.37

282

(1H, dd, J = 16.2, 7.8 Hz, Ha) and H 3.28 (1H, dd, J = 16.2, 9.6 Hz, Hb), an

283

oxymethine proton at H 4.90 (1H, dd, J = 9.6, 7.8 Hz), and two methyl signals at H

284

1.30

285

13-dimethyl-7,8-dihydrofurocoumarin skeleton (columbianetin).24,25 An anomeric

(3H,

s)

and

H

1.15

(3H,

s)

were

14


consistent

with

the

Page 15 of 38


286

signal at H 4.46 (1H, d, J = 7.8 Hz) / C 97.1 indicated that 1 is a monoglycoside. The

287

sugar unit for compound 1 was identified to be glucose according to its

288

data.20 In addition, the remaining

289

(CH2), 45.3 (CH2), 27.4 (CH3) and 68.8 (C) indicated that 1 contains a

290

3-hydroxy-3-methylglutaryl (HMG) moiety.20 Key HMBC correlations from H-6

291

[4.32 (1H, dd, J = 11.4, 1.8 Hz, Ha) and 3.98 (1H, dd, J = 11.4, 7.8 Hz, Hb)] to C-1

292

(δC 170.4) located the HMG moiety at C-6 of glucose (Figure 2). The negative optical

293

rotation value {[] D -55.2 (c 0.5, CH3OH)} of the aglycone, measured after

294

hydrolysis, indicated that 1 had an R configuration at C-12.26,27 The D-configuration

295

of the glucosyl moiety was identified by acid hydrolysis and chemical derivatization

296

of the released sugar.19 The anomeric proton of

297

β-orientation by the large coupling constant of 7.8 Hz. The S configuration of C-3 of

298

HMG moiety was confirmed by comparison of the optical rotation value {[α] D -6.2 (c

299

0.5, EtOH)} of 1-β-phenylethyl-mevalonamide, which was acquired by introduction

300

of β-phenylethylamine at the C-5″ of HMG moiety and subsequent hydrolysis by

301

LiBH4, with reference data.28 Therefore, the structure of compound 1 was determined

302

as (3″S, 12R) 6-O-3-hydroxy-3-methylglutaryl-columbianoside and named as

303

columbianoside Ⅰ. The 1H- and 13C-NMR data were assigned by 1D- and 2D-NMR

304

spectra (Table 1).

13C

13C-NMR

resonance at δC 170.4 (C), 172.3 (C), 45.2

27

D-glucose

was identified as

27

305

Compound 2 was isolated as yellow amorphous solid. The HR-ESI-MS

306

displayed a quasimolecular ion at 553.1928 [M + H]+, suggesting that it has a

307

molecular formula of C26H32O13, the same as that of compound 1. The similar UV and 15



Page 16 of 38

308

IR spectra of 2 with 1 suggested that they have the similar coumarin core. Comparison

309

of the 1H- and 13C-NMR data of 2 with 1 suggested that it has the great similarity with

310

compound 1 and their difference lay in the connection location between the glucose

311

and HMG moieties. The HMBC correlation from H-3′ [δH 4.95 (1H, t, J = 9.6 Hz)] to

312

C-1″ (δC 172.4) confirmed that the HMG moiety was linked to C-3′ of glucose (Figure

313

2). The D-configuration of the glucosyl moiety was identified by acid hydrolysis and

314

chemical derivatization of the released sugars. Based on the coupling constant of the

315

anomeric protons [H 4.68 (1H, d, J = 7.8 Hz)], the sugar moiety of 2 was

316

characterized as β-D-glucose. The negative optical rotation value of the aglycone

317

indicated that 2 had an R configuration at C-12. The configuration of HMG moiety

318

has not been determined due to its low amount. Thus, compound 2 was identified as

319

(12R)

320

columbianoside Ⅱ. Assignment of the 1H- and

321

and 2D-NMR experiments (Table 1).

3-O-3-hydroxy-3-methylglutaryl-columbianoside 13C-NMR

and

named

as

data was achieved by 1D-

322

Compound 4 has a molecular formula of C15H18O6 from the quasimolecular ion

323

at m/z 317.1004 [M + Na]+ (calcd for C15H18O6Na, 317.1001) in the HR-ESI-MS

324

together with the 13C-NMR data, accounting for seven degrees of unsaturation. The IR

325

absorptions displayed the presence of hydroxy functionality at 3383 cm-1 and carbonyl

326

functionality at 1693 cm-1. The UV spectrum showed the presence of a coumarin

327

skeleton core (λmax 208, 262, 324 nm).29 The 1H-NMR data (Table 1) displayed one

328

pair of characteristic doublets for H-3 [H 6.09 (1H, d, J = 9.6 Hz)] and H-4 [H 8.12

329

(1H, d, J = 9.6 Hz)], an aromatic singlet signal at H 6.44 (1H, s, H-6), a methoxy 16


Page 17 of 38


330

singlet at 3.87 (3H, s, 7-OCH3), and a 2, 3-dihydroxy-3-dimethylbutyl group at H

331

2.92 (2H, m, H-11), H 3.60 (1H, dd, J = 7.4, 5.5 Hz, H-12), H 1.27 (3H, s, H-14),

332

and H 1.24 (3H, s, H-15). The 1H- and 13C-NMR data of 4 were comparable to those

333

of meranzin hydrate (10), displaying the absence of a methine (-CH) proton at δH 7.55

334

(1H, d, J = 8.7 Hz, H-5) in 10.30,31 The 13C NMR data showed the deshielding effect

335

of C-5 from δC 126.9 (10) to δC 155.9 (4), indicating hydroxyl group substitution at

336

C-5 of 4, which was consistent with the molecular formula data. The HMBC

337

correlations from H-11 [H 2.92 (2H, m)] and H-12 [H 3.60 (1H, dd, J = 7.4, 5.5 Hz)]

338

to C-8 (C 107.8) indicated that the 2, 3-dihydroxy-3-dimethylbutyl group was

339

attached to C-8 of 4.

340

(C 163.4) located the -OCH3 group at C-7. The assignments of 1H- and

341

data were accomplished by 1H,

342

2). The negative optical rotation value {[α] D -40.5 (c 0.5, CH3OH)} indicated that 4

343

had an S configuration at C-12.30 Consequently, compound 4 was identified as

344

(12S)-5-hydroxyl-meranzin hydrate and named as meranzin hydrate Ⅰ.

The HMBC correlation from 7-OCH3 [H 3.87 (3H, s)] to C-7

13C

13C-NMR

NMR, 1H-1H COSY and HMBC spectra (Figure 29

Compound 5 was obtained as a yellow colloidal solid. The molecular formula

345

13C-NMR

346

was identified to be C15H18O6 using

spectrum and HR-ESI-MS data (m/z

347

317.1002 [M + Na]+, calcd for C15H18O6Na, 317.1001), the same as that of 4. The

348

1H-NMR

349

of aromatic doublets at H 7.31 (1H, d, J = 8.5 Hz, H-5) and H 6.99 (1H, d, J = 8.5

350

Hz,

351

3-dihydroxy-3-dimethylbutyl group at H 3.04 (2H, m, H-11), H 3.67 (1H, dd, J =

data (Table 2) exhibited a singlet signal for H-4 [H 7.02 (1H, s)], one pair

H-6),

a

methoxy

singlet

at

3.90

(3H,

17


s,

7-OCH3),

and

a

2,


352

9.2, 3.7 Hz, H-12), H 1.28 (3H, s, H-14), and H 1.30 (3H, s, H-15). The 1H NMR

353

spectrum of 5 showed the presence of a methine (-CH) proton at δH 7.31 (1H, d, J =

354

8.5 Hz, H-5) and the absence of a methine (-CH) proton for H-3 at δH 6.09 (1H, d, J =

355

9.6 Hz) compared with those of 4. The deshielding of C-3 resonance from δC 109.9

356

(4) to δC 140.3 (5) located the hydroxy group at C-3 for 5. The assignments of 1H- and

357

13C-NMR

358

The stereochemistry of C-12 was identified to be S by the negative optical rotation

359

value {[] D

360

(12S)-3-hydroxyl-meranzin hydrate and named as meranzin hydrate Ⅱ.

data were achieved by 1H-1H COSY and HMBC correlations (Figure 2).

29

-22.2 (c 0.5, CH3OH)}.30 Therefore, 5 was characterized as

361

Compound 6 was obtained as a yellow, amorphous solid. The HR-ESI-MS of 6

362

showed a quasimolecular ion at m/z 505.1692, which, in conjunction with the

363

13C-NMR

364

1) exhibited one pair of characteristic doublets for H-3 [H 6.26 (1H, d, J = 9.5 Hz)]

365

and H-4 [H 7.97 (1H, d, J = 9.5 Hz)], one pair of aromatic doublets signal at H 7.55

366

(1H, d, J = 8.6 Hz, H-5) and H 7.05 (1H, d, J = 8.6 Hz, H-6), a methoxy singlet at H

367

3.89 (3H, s, 7-OCH3), and a 2, 3-dihydroxy-3-dimethylbutyl group at H 2.90 (1H, dd,

368

J = 13.3, 10.1 Hz, H-11a), 2.73 (1H, dd, J = 13.0, 3.2 Hz, H-11b), 3.68 (1H, ddd, J =

369

10.1, 4.6, 2.5 Hz, H-12), and H 1.25 (6H, s, H-14 and H-15), which indicated the

370

presence of meranzin hydrate unit.31 An anomeric signal at H 4.43 (1H, d, J = 7.8 Hz)

371

/ C 97.0 indicated the presence of a sugar unit, which was identified as glucose by

372

analyses of the 1H- and

373

determined by acid hydrolysis and chemical derivatization of the released sugar. The

data, indicated a molecular formula of C23H30O11. The 1H-NMR data (Table

13C-NMR

data.

D-Configuration

18


of the glucosyl unit was

Page 18 of 38

Page 19 of 38


374

anomeric proton of

D-glucose

was identified as β orientation from the coupling

375

constant of 7.8 Hz. In addition, the remaining methyl signal at H 1.76 (3H, s) / C

376

20.2 and carbonyl signal at C 170.1 indicated the presence of an acetyl unit. The

377

HMBC correlations of H-6' [H 4.21 (1H, dd, J = 11.6, 2.2 Hz, 6'-Ha) / 4.00 (1H, dd, J

378

= 11.6, 8.1 Hz, 6'-Hb)] with CH3-CO- (C 170.1), and H-1' [H 4.43 (1H, d, J = 7.8

379

Hz)] with C-13 (C 80.1) located the acetyl unit at C-6´ of the glucosyl moiety and the

380

sugar unit at C-13 of the aglycone, respectively. The R configuration of C-12 was

381

determined by acid hydrolysis and comparison of the aglycone optical rotation value

382

{[α] D 27.06 (c 0.5, CH3OH)} with (-)-meranzin hydrate.32,33 Thus, 6 was identified as

383

(12R)-13-O-β-D-glucosyl-meranzin hydrate and named as meranzin hydrate Ⅲ.

29

384

Compound 7, obtained as a yellow amorphous solid, has the molecular formula

385

C21H28O69 from 13C-NMR and HR-ESI-MS data (m/z 447.1631 [M + Na]+, calcd for

386

C21H28O69Na, 447.1631). The 1H-NMR spectrum showed characteristic signals of a

387

coumarin skeleton, one pair of characteristic doublets for H-3 [H 6.23 (1H, d, J = 9.4

388

Hz)] and H-4 [H 7.89 (1H, d, J = 9.4 Hz)], one pair of aromatic doublets signal for

389

H-5 [H 7.50 (1H, d, J = 8.7 Hz)] and H-6 [H 7.04 (1H, d, J = 8.7 Hz)]. Comparison

390

of the 1H- and 13C-NMR signals with those of 7-methoxy-8-(2-formyl-2-methypropyl)

391

coumarin (paniculin I)34 revealed that 7 lacked a formyl group but possessed a

392

hydroxymethyl group [δH 3.25 (1H, d, J = 9.3 Hz) and 3.80 (1H d, J = 9.3 Hz) / δC

393

80.4] at C-12 of the aglycone. The aglycone of 7 was identified as

394

7-methoxy-8-(2-hydroxymethyl-2-methypropyl) coumarin by analyses of the 1H-1H

395

COSY, HSQC, and HMBC spectra. An anomeric signal at H 4.25 (1H, d, J = 7.8 Hz) 19



Page 20 of 38

396

/ C 105.1 indicated the presence of a sugar unit, which was concluded to be

397

β-D-glucose by analyses of the 1H- and

398

hydrolysis and chemical derivatization of the released sugar. The HMBC correction

399

from H-1' [δH 4.25 (1H, d, J = 7.8 Hz)] to C-13 (δC 80.4) located the sugar moiety at

400

C-13

401

13-O-β-D-glucosyl-7-methoxy-8-(2-hydroxymethyl-2-methypropyl)

402

named as paniculin Ⅲ.

403

of

the

aglycone.

Thus,

13C-NMR

compound

data in combination with acid

7

was

identified

to

coumarin

be and

Compound 8 was obtained as a yellow amorphous solid and had a molecular 13C-NMR

404

formula of C20H26O9 by analyses of

405

[M + Na]+, calcd for C20H26O9Na, 433.1475). The 1H- and

406

compound 8 suggested that it had the same aglycone as that of 6. An anomeric signal

407

at H 4.31 (1H, d, J = 4.8 Hz) / C 103.4 indicated the presence of a sugar unit, which

408

was concluded to be arabinofuranose from analyses of the 1H- and

409

resonances.35 L-Configuration of the arabinofuranosyl moiety was identified by acid

410

hydrolysis and chemical derivatization of the resulting sugar. The HMBC correlation

411

of H-1' [H 4.31 (1H, d, J = 4.8 Hz)] with C 87.3 (C-12) located the sugar moiety at

412

C-12 of aglycone. The R configuration of C-12 was determined by comparison of the

413

optical rotation value {[α] D 23.3 (c 0.5, CH3OH)} of the aglycone with (-)-meranzin

414

hydrate after acid hydrolysis.31,32 Therefore, compound 8 was identified as

415

(12R)-12-O-L-arabinofuranosyl-meranzin hydrate and named as meranzin hydrate Ⅳ.

416 417

and HR-ESI-MS data (m/z 433.1482 13C-NMR

data of

13C-NMR

29

The 10 known compounds were identified to be columbianoside (3)36, mexoticin (9)30,

meranzin

hydrate

(10)37,

auraptenol

(11)38,

20


isomeranzin

(12)39,

Page 21 of 38


418

8-[3-(β-D-glucopyranosyloxy)-2-hydroxy-3-methylbutyl]-7-methoxy-2H-1-benzopyra

419

n-2-one (13)36, praeroside VI (14)40, 5,7-dihydroxycoumarin (15)36, oxypeucedanin

420

hydrate (16)41, and bergaptol (17)42. The chemical structures of these compounds were

421

identified by comparing their physical and spectroscopic data with reported data.

422 423

Hepatoprotective activities of coumarins 1-17. To determine if these

424

compounds could show any hepatoprotective activities, we measured effects of

425

compounds 1-17 against

426

cells by the MTT assay. Bicyclol, a member of the latest generation of anti-hepatitis

427

drug with the hepatoprotective mechanisms involving the clearance of ROS,

428

regulation of cytokine expression, and inhibition of apoptosis induced by

429

immunological injury, was used as positive control.43 The results are summarized in

430

Table 3, which showed that compounds 3-5 and 17 exhibited strong hepatoprotective

431

activities with the inhibition values on

432

reduction of 49.0%, 36.5 %, 42.6% and 32.6%, respectively. Interestingly, the

433

protective effects of these compounds were even greater than that of bicyclol. In

434

contrast, the remaining compounds showed little to no hepatoprotective activities.

D-galactosamine-induced

cell survival inhibition in LO2


cell survival

435

Structure-activity relationship (SAR) analysis revealed that the introduction of

436

HMG group and acetyl group at the sugar moiety of coumarin decreases its

437

hepatoprotective effect since compounds 1 and 2 showed less hepatoprotective effects

438

than 3, and compound 6 showed less hepatoprotective effect than 13. The introduction

439

of hydroxyl group at coumarin core increased the hepatoprotective effects of 21



440

coumarins due to the higher hepatoprotective effects of compounds 4, 5 and 17.

441

However, the methylation of hydroxyl group will attenuate the hepatoprotective

442

effects as compound 9 showed poor hepatoprotective effect. By assessing two potent

443

compounds 3 and 4, we found that coumarins suppressed the growth inhibitory effect

444

of

445

statistical significance was observed among these doses. In the following studies, we

446

focused on compounds 3 and 4 as they are the tops hits that elicited the strongest

447

protective effects.

D-galactosamine

in a relatively wide range of doses (Figure S59), although no

448 449

Morphological changes of LO2 cells. The hepatotoxicity of D-galactosamine

450

was further evaluated by monitoring the cell morphology. The results show that

451

treatment of LO2 cells with 40 mM D-galactosamine for 24 h resulted in dramatic

452

cellular morphology changes. Most cells lost neurites and some of which were lysed

453

or replaced by debris (Figure 3). In contrast, culture exposure to the same amount of

454

D-galactosamine

455

cell morphology, similar to the positive control bicyclol (Figure 3). These results

456

suggest that compounds 3 and 4 elicited protective effects against D-galactosamine

457

induced liver cell injury.

in the presence of compounds 3 or 4 remarkably preserved normal

458 459

Effects of compounds 3 and 4 on ALT and AST activities. ALT and AST are

460

two key liver enzymes, whose levels reflect the health of the liver and hepatocyte

461

integrity. Increases in these enzymes’ activities may be associated with a decrease in 22


Page 22 of 38

Page 23 of 38


462

liver functional mass, which ultimately leads to hepatopathy.44 Therefore, these two

463

enzymes are widely considered as biomarkers of liver function. To further determine

464

the hepatoprotective function of coumarins, we measured levels of ALT and AST

465

activities in injured LO2 cells. Treatment with

466

elevated the ALT level and, to a lesser extent, the AST level in LO2 cells (Figure 4A).

467

However, pretreatment of cells with compound 3 or 4 greatly suppressed the increases

468

in ALT and AST levels induced by D-galactosamine (Figure 4A), further supporting

469

that compounds 3 and 4 have hepatoprotective effects against

470

induced liver cell injury. Interestingly, the protective effects of compounds 3 and 4

471

were greater than that of the positive control bicyclol (Figure 4A), indicating that

472

these compounds could be developed into potent hepatoprotective agents.

D-galactosamine

alone markedly

D-galactosamine

473 474

Determination of the activities of SOD and GSH-Px and the content of MDA.

475

D-Galactosamine

has been used as an experimental model to mimic hepatitis-induced

476

liver injury.45-47 The mechanism of

477

believed to be through ATP depletion, which finally results in hepatocyte necrosis. D-

478

Galactosamine provokes a state of oxidative stress by releasing free radicals and

479

reducing antioxidant enzymes.45 The toxicity of

480

accompanied with increased lipid peroxides.46 To determine the protective

481

mechanisms of compounds 3 and 4 against

482

damage, we investigated their effects on the activities of antioxidant enzymes. In the

483

antioxidant defenses system, SOD is involved in the direct elimination of ROS; on the

D-galactosamine

induced hepatotoxicity is

D-galactosamine


23


is often

liver cell


Page 24 of 38

484

other hand, GSH is the most abundant low-molecular weight endogenous antioxidant

485

in maintaining the integrity of cells.47 In addition, intracellular content of MDA, a

486

product of lipid peroxidation, always reflects the degree of lipid peroxidation

487

damage.45 We found that D-galactosamine reduced the activities of GSH-Px and SOD

488

by 18.7 % and 80.3 %, respectively (Figure 4B-C). In contract, it increased the MDA

489

level by 1.3 folds in LO2 cells (Figure 4D). Treatment with compound 3 or 4

490

significantly attenuated the decreases in GSH-Px and SOD activities caused by

491

D-galactosamine

492

level in LO2 cells although the difference was not statistically significant (Figure 4D).

493

These effects of compounds 3 and 4 were greater than that of bicyclol except the

494

reduction in the MDA level (Figure 4B-D). Together, these results strongly support

495

the hepatoprotective effects of compounds 3 and 4 and suggest that these compounds

496

protect liver cells from

497

anti-oxidative properties.

(Figure 4B-C). Compounds 3 and 4 also reduced the increased MDA


injury likely through their

498 499

In summary, here we report the isolation and identification of seven new

500

coumarin compounds, together with ten known analogues, from the air-dried pericarp

501

of Citrus grandis. We further characterized the hepatoprotective activities of these

502

compounds,

503


504

mechanisms underlying the protective effects of coumarins are through modulating

505

the cellular antioxidant pathway. These results reveal the importance of coumarins for

especially

the

two

potent

compounds

3

and

4,

against

LO2 liver cell injury. We subsequently reveal that the

24


Page 25 of 38


506

Citrus grandis in the hepatoprotective function, which may broaden the application

507

and accelerate the development of Citrus grandis as a potent hepatoprotective agent.

508 509

■ ASSOCIATED CONTENT

510

Supporting Information

511

The Supporting Information (Figures S1-S59) is available free of charge on the ACS

512

Publications website

513 514

■ AUTHOR INFORMATION

515

Corresponding Authors

516

*(X.-S. Yao) E-mail: [email protected].

517

*(J.-S. Tang) E-mail: [email protected]

518

ORCID

519

Jinshan Tang:

520

Author Contributions

521

‖D.M.

522

Notes

523

The authors declare no competing financial interest

Tian and F.F. Wang contributed equally to this work

524 525

■ ACKNOWLEDGMENTS

526

We are grateful to Ms. P. Lin, Ms. W.J. Yun, Mr. Y.C. Ren, Mr. J. Qiao and Dr. Y.

527

Yu for the HR-ESI-MS and NMR measurements. Part of the chemical work was 25



528

accomplished in Guangzhou Xiangxue Pharmaceutical Ltd., Co. This work was

529

supported by grants from Science and Technology Planning Project of Guangdong

530

Province, China (No.2015B030301005).

531 532

■ REFERENCES

533

(1) Duan, L.; Guo, L.; Dou, L. L.; Yu, K. Y.; Liu, E H.; Li, P. Comparison of

534

chemical profiling and antioxidant activities of fruits, leaves, branches, and flowers of

535

Citrus grandis 'Tomentosa'. J. Agr. Food Chem. 2014, 62, 11122-11129.

536

(2) The State Pharmacopoeia Commission of PRC, Citri Grandis Exocarpium

537

(Huajuhong), Pharmacopoeia of the People's Republic of China, China Medical

538

Science and Technology Press, Beijing, China, 2015, 1, 74-75.

539

(3) Zhang, M.; Duan, C.; Zang, Y.; Huang, Z.; Liu, G. The flavonoid composition of

540

flavedo and juice from the pummelo cultivar (Citrus grandis (L.) Osbeck) and the

541

grapefruit cultivar (Citrus paradisi) from China. Food Chem. 2011, 129, 1530-1536.

542

(4) Tsai, H. L.; Chang, S. K. C.; Chang, S. J. Antioxidant content and free radical

543

scavenging ability of fresh red Pummelo [Citrus grandis (L.) Osbeck] juice and

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freeze-dried products. J. Agr. Food Chem. 2007, 55, 2867-2872.

545

(5) Wen, B.; Cai, C. T.; Wang, R. L.; Tan, Y. H.; Lan, Q. Y. Critical moisture content

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windows differ for the cryopreservation of pomelo (Citrus Grandis) seeds and

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embryonicaxes. Cryoletters 2010, 31, 29-39.

548

(6) Wu, T. S.; Huang, S. C.; Jong, T. T.; Lai, J. S.; Kuoh, C. S. Coumarins, acridone

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alkaloids and a flavone from Citrus grandis. Phytochemistry 1988, 27, 585-587. 26


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(7) Wu, T. S.; Huang, S. C.; Lai, J. S. Stem bark coumarins of Citrus grandis.

551

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Citrus grandis. Phytochemistry 1989, 28, 3574-3576.

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Citrus grandis buntan. Phytochemistry 1996, 42, 221-223.

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of aldose enantiomers by reversed-phase HPLC. Chem. Pharm. Bull. 2007, 55,

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inhibitory effects on the H1N1 influenza virus. J. Nat. Prod. 2017, 80, 2818-2824.

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of the acyl group of 8'-O-(3-hydroxy-3-methylglutaryl)-8'-hydroxyabscisic acid.

592


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(22) Liu, Y. F.; Liang, D.; Luo, H.; Hao, Z. Y.; Wang, Y.; Zhang, C. L.; Zhang, Q. J.; 28


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Rehmannia glutinosa. J. Nat. Prod. 2012, 75, 1625-1631.

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(27) Chen, Y. H.; Chang, F. R.; Wu, C. C.; Yen, M. H.; Liaw, C. C.; Huang, H. C.;

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hedyotiscone A - C, from Hedyotis biflora. Planta Med. 2006, 72, 75-78.

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612

of the acyl group of 8'-O-(3-hydroxy-3-methylglutaryl)-8'-hydroxyabscisic acid.

613


614

(29) Liu, B. Y.; Zhang, C.; Zeng, K. W.; Li, J.; Guo, X. Y.; Zhao, M. B.; Tu, P. F.;

615

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and stems of Triphasia trifolia. Fitoterapia 2006, 77, 129-133.

619

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identification of metabolites of osthole in rats. Xenobiotica 2012, 42, 1120-1127.

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determination of chiral alkaloids and coumarins derived from O- and C-prenyl

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Coumarins from Leonurus japonicus and their anti-platelet aggregative activity.

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Chinese drug Qianhu, XXVII: structure of a new simple coumarin glycoside from

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(41) Zhao, A. H.; Yang, X. W.; Yang, X. B.; Liu, J. X.; Wang, Q. L.; Wang, W. Q. A

649

new natural product from root of Angelica dahurica cv. Qibaizhi. Zhongguo

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659

Active fragment of Veronica ciliata fisch. attenuates t-BHP-induced oxidative stress 31



Page 32 of 38

660

injury in HepG2 cells through antioxidant and antiapoptosis activities. Oxid. Med.

661

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662

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664

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665

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666

Hepatoprotective effects of nicotiflorin from Nymphaea candida against Concanavalin

667

A-induced and D-Galactosamine-induced liver injury in mice. Int. J. Mol. Sci. 2017,

668

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669

(47) Jiang, M. Z.; Yan, H.; Wen, Y.; Li, X. M. In vitro and in vivo studies of

670

antioxidant activities of flavonoids from Adiantum capillus-veneris L, Afr. J. Pharm.

671

Pharmaco. 2011, 5, 2079-2085.

672 R2

O R 3O

O

R 4O

5 4 10 4 (12S) R1= R3=R4=H, R2=OH 6 R1 5 (12S) R =OH R = R = R =H 1 2 3 4 7 6 (12R) R1= R2= R3=H R4=6'-O-acetyl--D-glc O O 89 O 8 (12R) R1=R2= R4=H, R3=L-ara 12 11 14 9 (12S) R1= R3= R4= H, R2= OCH3 13 10 (12S) R1= R2= R3= R4=H RO 15 13 (12S) R1= R2= R3=H R4=-D-glc 14 (12S) R1= R2= R4= H, R3=-D-glc

3 2 O 1 R = 6'-O-HMG--D-glc 2 R = 3'-O-HMG--D-glc 3 R = -D-glc

OH O

O

O

O HO

O

O

O

O O

O

11

OR

12 O

HO

RO 7 R=-D-glc

11

12

Figure 1. Chemical structures of compounds 1-17.

32


15

O

O

O

O

4 HO 1 2 16 R = 5 3 OH 17 R = H

Page 33 of 38


O HO

O

O

O O

O HO HO

O O OH 6

O O

O HO

OH

O

O

OH

OH

HO

2

O

O HO

O

O

OH 5

4

O O

HO HO HO

OH

O

OH

O

O

OH

HO HO O

1

O HO

O

O O

HO HO

O

O

O OH

O

O HO

O O OH

O OH

O O

O

O

OH

1

OH

7

H-1H COSY HMBC

8

Figure 2. Key 1H-1H COSY and HMBC correlations of compounds 1, 2, 4-8.

A

B

D

E

C

Figure 3. Effects of coumarins 3 and 4 on LO2 cell morphology induced by D-galactosamine. (A) LO2 cells were treated with DMSO. (B) LO2 cells were exposed to 40 mM D-galactosamine for 24 h. There is a significant decrease in cell number. (C&D&E) LO2 cells were preincubated with 20 μM bicyclol, compounds 3 and 4, respectively, and then exposed to 40 mM D-galactosamine for 24 h. Scale bar, 50 μm.

33



A

Liver function index

##

# *

* *

*

4

SOD (U/mgprot)

B Control 6 D-GalN D-GalN+Bicyclol D-GalN+3 D-GalN+4

8 6

4

**

2

*

##

2

+4 -G al N

D

D

-G al N

+3

cl ol ic y

-G al N D

-G al N

D 20

+B

C

D

AST (U/L)

C

ALT (U/L)

on tr ol

0

0

1.0

## 15

*

#

MDA (nmol/mgprot)

*

10 5

* 0.6 0.4 0.2

+4 D

-G al N

+3

D

-G al N

D

-G al N

lo l yc ic +B

C

-G al N

on tr ol

+4 -G al N

+3 D

-G al N

D

-G al N

D

+B

ic

yc

-G al N D

co

lo l

0.0 nt ro l

0

0.8

D

GSH-Px (mU/mgprot)

Page 34 of 38

Figure 4. Effects of coumarins 3 and 4 on alanine transaminase (ALT) and aspartate transaminase (AST), antioxidant enzymes and malondialdehyde (MDA) levels. ALT and AST activities (A), superoxide dismutase (SOD) activities (B), glutathione peroxidase (GSH-Px) activities (C), MDA levels (D) in LO2 cells. Cells were incubated with 40 mM D-galactosamine alone for 24 h or were pretreated with coumarin 3 or 4 for 4 h prior to D-galactosamine addition. Values are mean ± S.E.M. and at least two independent experiments were carried out in triplicates. #p < 0.05, ##p < 0.01 compared with the control group. *p < 0.05, **p < 0.01 compared with the D-GalN group.

O HO

OH S

O O

O

O

O

HO HO

O

O a

N H

O

b

5'' 6''

1''

O 2'

1'

N H

O

O

O

O

OH A

3'' 2''

O

O HO HO

OH 1

4''

OH O

1

OH 2

3

R

4

5

OH

B

Scheme 1. Determination of the absolute configuration of the 3-hydroxy-3-methylglutaric

acid (HMG) moiety in compound 1. Reaction condition and reagent: (a) β-phenylethylamine, PyBOP, HOBt, Et3N, r.t., 9h; (b) LiBH4, THF, r.t., 24h. PyBOP: (benzotriazol-1-yloxy) 34


Page 35 of 38


tripyrrolidinophosphonium hexafluorophosphate; HOBt: 1-hydroxybenzotriazole; DMF: N, N-dimethylformamide; THF: tetrahydrofuran.

Table 1. 1H- and 13C-NMR Spectroscopic Data of Compounds 1, 2 and 4. No.

1a

C, type

2b

H (J in Hz)

C, type

4b

H (J in Hz)

C, type

163.2, C

H (J in Hz)

2

160.1, C

3

111.5, CH

6.23, d (9.5)

112.3, CH

6.18, d (9.5)

109.9, CH

6.09, d (9.6)

4

144.8, CH

7.96, d (9.5)

146.3, CH

7.85, d (9.5)

141.7, CH

8.12, d (9.6)

5

129.1, CH

7.48, d (8.6)

130.3, CH

7.40, d (8.3)

155.9, C

6

106.4, CH

6.82, d (8.6)

107.9, CH

6.76, d (8.3)

95.2, CH

7

163.4, C

165.6, C

163.4, C

8

113.6, C

115.3, C

107.9, C

9

150.8, C

152.6, C

155.3, C

10

112.7, C

114.5, C

104.2, C

11

27.0, CH2

12

89.7, CH

13

77.2, C

14

20.2, CH3

1.15, s

21.4, CH3

1.28, s

25.5, CH3

1.27, s

15

23.5, CH3

1.30, s

23.7, CH3

1.43, s

25.5, CH3

1.24, s

56.4, CH3

3.87, s

3.37, dd (16.2, 7.8) Ha 3.28, dd (16.2, 9.6) Hb 4.90, dd (9.6, 7.8)

164.4, C

28.5, CH2 91.6, CH

3.44, dd (16.2, 7.8) Ha 25.8, CH2

5.00, dd (9.6, 7.8)

1'

97.1, CH

4.46, d (7.8)

98.7, CH

4.68, d (7.8)

2'

73.3, CH

2.93, m

73.3, CH

3.28, dd (9.6,7.8)

3'

76.5, CH

3.16, m

79.2, CH

4.95, t (9.6)

4'

70.3, CH

3.04, m

69.8, CH

3.47, m

5'

73.4, CH

3.38, m

77.6, CH

3.37, m

6'

63.7, CH2

1''

170.4, C

2''

45.2, CH2

3''

68.8, C

4''

45.3, CH2

5''

172.3, C

6''

27.4, CH3

aMeasured

in DMSO-d6

79.1, CH

62.6, CH2

3.85, dd (11.9, 2.2) Ha 3.67, dd (11.7, 5.7) Hb

172.4, C 2.60, m

47.0, CH2

2.74, m

71.0, C 2.49, m

45.9, CH2

2.64, m

175.8, C 1.21, s bMeasured

3.60, dd (7.4, 5.5)

74.1, C

7-OCH3

3.98, dd (11.4, 7.8) Hb

2.92, m

3.39, dd (16.2, 9.6) Hb

79.5, C

4.32, dd (11.4, 1.8) Ha

6.44, s

27.8, CH3

1.40, s

in CD3OD

Multiplets and or overlapped signals are reported without designating multiplicity. 35



Page 36 of 38

Table 2. 1H- and 13C-NMR Spectroscopic Data of Compounds 5-8 (in CD3OD). No.

5

C, type

6

H (J in Hz)

C, type

7

H (J in Hz)

C, type

8

H (J in Hz)

H (J in Hz)

2

161.5, C

160.4, C

3

140.3, C

112.1, CH

6.26, d (9.5)

112.9, CH

6.23, d (9.4)

113.5, CH

6.25, d (9.4)

4

117.5, CH

144.7, CH

7.97, d (9.5)

146.5, CH

7.89, d (9.4)

146.1, CH

7.88, d (9.4)

5

125.9, CH

7.31, d (8.5) 127.0, CH

7.55, d (8.6)

128.6, CH

7.50, d (8.7)

128.9, CH

7.54, d (8.7)

6

109.3, CH

6.99, d (8.5) 107.9, CH

7.05, d (8.6)

109.0, CH

7.04, d (8.7)

108.9, CH

7.05, d (8.7)

7

159.5, C

160.4, C

162.9, C

162.1, C

8

116.8, C

115.7, C

116.7, C

115.6, C

9

150.3, C

153.0, C

154.8, C

154.6, C

10

115.6, C

112.5, C

114.2, C

114.5, C

11

26.5, CH2

7.02, s

3.04, m

163.7, C

C, type 163.4, C

24.7, CH2 2.90, dd (13.3, 10.1) 32.0, CH2 2.92, d (13.2) Ha 26.4, CH2 3.17, dd (13.7, 9.7) Ha Ha

2.88, d (13.2) Hb

2.96, dd (13.7, 3.2) Hb

2.73, dd (13.0, 3.2) Hb 12

78.9, CH

3.67, dd (9.2, 75.2, CH 3.68, ddd (10.1, 4.6, 3.7)

13

74.1, C

38.4, C

87.3, CH

3.91, dd (9.7,3.2)

2.5) 80.1, C

80.4, CH2 3.80, d (9.3) Ha

73.7, C

3.25, d (9.3) Hb 14

25.6, CH3

1.28, s

23.4, CH3

1.25, s

25.3, CH3

0.95, s

23.6, CH3

1.31, s

15

25.5, CH3

1.30, s

20.5, CH3

1.25, s

24.8, CH3

0.91, s

27.0, CH3

1.29, s

7-OCH3

56.6, CH3

3.90, s

56.2, CH3

3.89, s

56.4, CH3

3.92, s

56.7, CH3

3.87, s

1'

97.0, CH

4.43, d (7.8)

105.1, CH

4.25, d (7.8)

103.4, CH

4.31, d (4.8)

2'

73.5, CH

2.96, t (8.4)

75.3, CH 3.22, dd (9.2,7.8)

78.5, CH

3.48, dd (7.7, 4.8)

3'

76.3, CH

3.19, t (8.9)

78.2, CH

3.37, t (8.9)

74.9, CH

4.01, t (7.2)

4'

70.5, CH

3.03, t (9.3)

71.7, CH

3.30, m

83.6, CH

3.56, m

5'

73.3, CH

3.35, t (2.0)

77.9, CH

3.25, m

62.0, CH2

3.65, m Ha 3.57, m Hb

6'

63.8, CH2 4.21, dd (11.6, 2.2) Ha 62.8, CH2 3.86, dd (11.9, 2.2) 4.00, dd (11.6, 8.1) Hb

Ha 3.69, dd (11.9, 5.6) Hb

CH3-CO-C6′

170.1, C

CH3-CO-C6′

20.2, CH3

1.76, s

Multiplets and or overlapped signals are reported without designating multiplicity.

36


Page 37 of 38


Table 3. Hepatoprotective Effects of Compounds (20 μM)a compound

cell survival rate (% of normal)

normal

100.0 ± 0.8

model

73.3 ± 2.2

bicyclol

77.6 ± 0.5*

16.2

2

73.3 ± 1.2

0.1

3

inhibition (% of model)

86.4 ±

2.3**

49.0

83.0 ±

2.6**

36.5

5

84.7 ±

3.5**

42.6

11

75.9 ± 0.7

4

10.0

12

78.0 ±

2.1*

17.7

13

80.1 ± 4.0*

25.6

14

80.1 ± 5.4*

25.6

15 16 17 aResults

80.8 ±

7.5**

28.3

79.3 ±

5.6*

22.6

82.0 ±

2.4**

32.6

were expressed as means ± S.E.M. (n = 3); bicyclol was used as a

positive control (20 μM). **p < 0.01. *p < 0.05.

37



Page 38 of 38

TOC Graphic Coumarin Analogues from the Citrus grandis (L.) Osbeck and Their Hepatoprotective Activity

O

O

O

D-galactosamine

HO O

HO HO

O

OH

Hepatoprotective activities

3

OH

O HO

Citrus grandis (L.) Osbeck

injure LO2 cells

O

SOD and GSH-Px activities

O

AST, ALT and MDA levels OH 4

38


Coumarin Analogues from the Citrus grandis (L.) Osbeck and Their

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