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New Flavonol Glucuronides from the Flower Buds of Syzygium aromaticum (Clove) Byeol Ryu, Hye Mi Kim, Jin Su Lee, Chan Kyu Lee, Jurdas Sezirahiga , Jeong-Hwa Woo , Jung-Hye Choi, and Dae Sik Jang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00337 • Publication Date (Web): 05 Apr 2016 Downloaded from http://pubs.acs.org on April 9, 2016

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Journal of Agricultural and Food Chemistry

1

New Flavonol Glucuronides from the Flower Buds of Syzygium aromaticum

2

(Clove)

3 4

Byeol Ryu,† Hye Mi Kim,‡ Jin Su Lee,† Chan Kyu Lee,‡ Jurdas Sezirahiga,‡ Jeong-Hwa Woo,†

5

Jung-Hye Choi,†,‡ and Dae Sik Jang*,†,‡

6 7

†

Department of Life and Nanopharmaceutical Sciences and ‡College of Pharmacy, Kyung Hee University, Seoul, 02447, Republic of Korea

8 9 10 11 12

*Author to whom correspondence should be addressed [Tel:+82-2-961-0719; Fax:+82-2-966-

13

3885; E-mail: [email protected]].

14 15

Running Head: New Flavonol Glucuronides from Clove

16 17

ACS Paragon Plus Environment


18

ABSTRACT: Repeated chromatography of the EtOAc-soluble fraction from the 70 % EtOH

19

extract of the flower buds of Syzygium aromaticum (clove) led to the isolation and

20

characterization of four new flavonol glucuronides, rhamnetin-3-O-β-D-glucuronide (1),

21

rhamnazin-3-O-β-D-glucuronide (2), rhamnazin-3-O-β-D-glucuronide-6″-methyl ester (3), and

22

rhamnocitrin-3-O-β-D-glucuronide-6″-methyl ester (4), together with fifteen flavonoids (5 - 19)

23

having previously known chemical structures. The structures of the new compounds 1-4 were

24

determined by interpretation of spectroscopic data, particularly by 1D- and 2D-NMR studies. Six

25

flavonoids (6, 7, 9, 14, 18, and 19) were isolated from the flower buds of S. aromaticum for the

26

first time in this study. The flavonoids were examined for their cytotoxicity against human

27

ovarian cancer cells (A2780) using MTT assays. Among the isolates, pachypodol (19) showed

28

the most potent cytotoxicity on A2780 cells with IC50 value of 8.02 µM.

29 30 31

KEYWORDS: Syzygium aromaticum, clove, flavonol glucuronides, cytotoxicity, ovarian cancer

2


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32


INTRODUCTION

33

Syzygium aromaticum L. (syn. Eugenia caryophyllata Thunb.) is an evergreen tree and a

34

member of the Myrtaceae family. The flower buds of S. aromaticum (clove) have been

35

commonly used as a spice for salads, pickles and curries, and as a constituent material for

36

traditional East Asian medicines. Cloves originated from Eastern Indonesia, and harvested

37

primarily in Indonesia, India, Madagascar, Zanzibar, Pakistan, Sri Lanka and Tanzania.1

38

Cloves represent one of the major vegetal sources of phenolic compounds such as phenolic

39

acids (gallic acid), flavonol glucosides, phenolic volatile oils (eugenol, acetyl eugenol) and

40

tannins which show the wide ranges of pharmacological effect such as anti-inflammatory,2

41

antioxidant,3 antimicrobial,4 antibacterial,5 antifungal,6 antiviral,7

42

antimutagenic.9

antinociceptive,8 and

43

In our ongoing project to search natural anti-tumor agents, a 70% EtOH extract of the flower

44

buds of S. aromaticum exhibited a significant inhibitory activity in a preliminary in vitro

45

screening against human ovarian cancer cells (A2780) using MTT assays. Repeated

46

chromatography of the EtOAc-soluble fraction from the 70 % EtOH extract of cloves led to the

47

isolation and characterization of four new flavonol glucuronides (1 - 4), together with fifteen

48

flavonoids (5 - 19) having previously known chemical structures. The structures of the new

49

compounds were determined by spectroscopic data interpretation including spectroscopic data

50

(1H-NMR, 13C-NMR, 2D-NMR, and MS) analyses. Thereafter the flavonoids were evaluated for

51

their cytotoxicity on human ovarian cancer cells (A2780). This paper describes the isolation and

52

structural elucidation of the isolates and their cytotoxicity in vitro.

53 54

MATERIALS AND METHODS

3



55

General Methods. Optical rotations were measured on a Jasco P-2000 polarimeter, using a

56

10-cm microcell. UV spectra were obtained on Spectramax M5 (Molecular Devices, Sunnyvale,

57

CA, USA). HR-Mass spectra were obtained using an LTQ-Orbitrap mass spectrometer (Thermo

58

scientific, Waltham, MA, USA). NMR spectra were obtained using a Varian 500 MHz and a

59

Bruker 400 MHz NMR spectrometers using TMS or solvent residues as internal standards and

60

chemical shifts were expressed as δ values. IR spectra were obtained using a Nicolet iS50 FT-IR

61

spectrometer (Thermo scientific, Waltham, MA, USA). TLC analyses were performed on Silica

62

gel 60 F254 (Merck) and RP-18 F254S (Merck) plates using 20% (v/v) H2SO4 reagent (Aldrich) to

63

visualize compounds. Silica gel (Merck 60A, 70-230 or 230-400 mesh ASTM), Sephadex LH-20

64

(Amersham Pharmacia Biotech), reversed-phase silica gel (YMC Co., ODS-A 12 nm S-150 µm),

65

LiChroprep RP-18 (Merck, 40-63 µm), and Diaion HP-20 (Mitsubishi) were used for column

66

chromatography. Pre-packed cartridges, Redi Sep-C18 (26 g, 43 g, Teledyne Isco) were used for

67

flash chromatography. Semi-preparative HPLC was performed using the Gilson Gastorr BG-34

68

degasser, Gilson 321 pump, Gilson UV/VIS-155 detector, with YMC Pack ODS-A column (250

69

× 200 mm i.d., 5 µm).

70

Plant Material. The flower buds of Syzygium aromaticum L. (Myrtaceae) were purchased

71

from a Kyungdong Crude Drugs Market (Seoul, South Korea), in June 2013. The origin of the

72

herbal material was identified by Prof. Dae Sik Jang. A voucher specimen (SYAR1-2013) has

73

been deposited at College of Pharmacy, Kyung Hee University, Republic of Korea.

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Extraction and Isolation. The dried and powdered plant material (2.1 kg) was extracted

75

with 20 L of 70 % EtOH twice at 60 °C in water bath for 2 h and the solvent was evaporated in

76

vacuo at 40 °C. The 70 % EtOH extract (625.0 g) was successively partitioned with n-hexane (2

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L × 3), EtOAc (2 L × 3), and BuOH (2 L × 3) to give n-hexane- (303.1 g), EtOAc- (132.0 g),

78

BuOH- (93.0 g), and water-soluble extracts (95.7 g), respectively.

79

The EtOAc-soluble extract (132.0 g) was fractionated into CH2Cl2-soluble and -insoluble

80

fractions. The CH2Cl2-soluble fraction (12.2 g) was chromatographed over silica gel (70-230

81

mesh; φ 6.0 × 44 cm) as stationary phase with a CH2Cl2-MeOH mixture (gradient from 1:0 to 9:1

82

v/v) as mobile phase to afford 7 fractions (EM1~EM7). Fraction EM3 [eluted with CH2Cl2-

83

MeOH (19:1 v/v); 7.2 g] was subjected to silica gel column chromatography (CC) (230-400

84

mesh, φ 5.0 × 33 cm, n-hexane-EtOAc = 4:1 to 1:1 v/v) to produce 8 subfractions (EM3-1~EM3-

85

8). Compounds 17 (17.8 mg), 18 (9.0 mg), and 19 (5.5 mg) were obtained from subfraction

86

EM3-4 by reversed-phase CC (YMC gel 150 µm, φ 3.5 × 24 cm) with MeOH-H2O mixture (7:3

87

v/v).

88

The

CH2Cl2-insoluble

fraction

(118.79

g)

of

the

EtOAc-soluble

extract

was

89

chromatographed over silica gel (70-230 mesh; φ 10.0 × 36 cm) as stationary phase with a

90

CH2Cl2-MeOH-H2O gradient (from 1:0:0 to 5:4.5:0.5 v/v/v) as mobile phase to afford 13

91

fractions (E1~E13). Fraction E3 [eluted with CH2Cl2-MeOH-H2O (9:1:0.1 v/v/v); 1.37 g] was

92

further subjected to silica gel CC (230-400 mesh, φ 5.0 × 33 cm, n-hexane-EtOAc-MeOH =

93

6:3.5:0.5 to 4:5:1 v/v/v) to produce 9 subfractions (E3-1~E3-9). Compound 16 (35.1 mg) was

94

obtained by recrystallization (MeOH) from subfraction E3-5. Compound 3 (22.0 mg) was

95

isolated from subfraction E3-8 by a medium pressed liquid chromatography (MPLC) with Redi

96

Sep-C18 (26 g, MeOH-H2O = 3:7 to 7:3 v/v) and reverse phase HPLC with an YMC Pack ODS-

97

A column. Subfraction E3-6 was further fractionated using a Sephadex LH-20 (φ 2.4 × 40 cm)

98

with CH2Cl2-MeOH mixture (1:1 v/v) to separate compound 15 (14.0 mg). Fraction E4 [eluted

99

with CH2Cl2-MeOH-H2O (9:1:0.1 v/v/v); 3.91 g] was further subjected to silica gel CC (230-400

5



100

mesh, φ 4.8 × 39 cm CH2Cl2-MeOH-H2O = 19:1:0.1 to 7:2.7:0.3 v/v/v) to produce 8 subfractions

101

(E4-1~E4-8). Subfraction E4-4 was subjected to a MPLC using Redi Sep-C18 (43 g, MeOH-

102

H2O, 2.5:7.5 to 8.5:1.5 v/v) cartridge to generate compound 4 (30.1 mg). Compound 13 (20.3

103

mg), 7 (8.0 mg), and 9 (29.4 mg) was purified from subfraction E4-5 by a flash chromatography

104

system using Redi Sep-C18 (26 g, MeOH-H2O, 3.5:6.5 to 7:3 v/v) cartridge. Compound 6 (9.9

105

mg) was purified from subfraction E4-7 by a reversed phase HPLC with an YMC Pack ODS-A

106

column. Fraction E5 [eluted with CH2Cl2-MeOH-H2O (9:1:0.1 v/v/v); 12.47 g] was subjected to

107

silica gel CC (230-400 mesh, φ 6.5 × 38 cm CH2Cl2-MeOH-H2O = 19:1:0.1 to 7:2.7:0.3 v/v/v) to

108

produce 6 subfractions (E5-1~E5-6). Subfraction E5-4 was further separated by CC using

109

LiChroprep RP-18 as stationary phase with MeOH-H2O (0:1 to 7:3 v/v) mixture to isolate

110

compounds 8 (21.0 mg), 10 (5.9 mg), and 12 (22.1 mg). Fraction E6 [eluted with CH2Cl2-

111

MeOH-H2O (8:1.8:0.2 v/v/v); 11.49 g] was subjected to silica gel CC (230-400 mesh, φ 5.8 × 40

112

cm, CH2Cl2-MeOH-H2O = 9:1:0.1 to 7:2.7:0.3 v/v/v) to produce 13 subfractions (E6-1~E6-13).

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Subfraction E6-10 was further separated by CC using LiChroprep RP-18 as stationary phase with

114

MeOH-H2O (0:1 to 7:3 v/v) mixture to give compounds 11 (322.8 mg). Compounds 14 (2.8 mg)

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and 2 (4.3 mg) were isolated from fraction E7 [eluted with CH2Cl2-MeOH-H2O (7:2.7:0.3 v/v/v);

116

2.5 g] by CC using LiChroprep RP-18 as stationary phase with MeOH-H2O (2:8 to 6:4 v/v)

117

mixture. Compounds 1 (3.8 mg) and 5 (23.4 mg) were obtained from fractions E9 [eluted with

118

CH2Cl2-MeOH-H2O (7:2.7:0.3 v/v/v); 470.9 mg] and E10 [eluted with CH2Cl2-MeOH-H2O

119

(6:3.6:0.4 v/v/v); 513.8 mg], respectively, by using a MPLC with Redi Sep-C18 (43 g, MeOH-

120

H2O = 3:7 to 7:3 v/v and 43 g, MeOH-H2O = 3:7 to 1:1 v/v, respectively).

121

Rhamnetin-3-O-β-D-glucuronide (1): yellowish amorphous powder; [ܽ]ଶ଴ ୈ : -10.56° (c 0.01,

122

MeOH); UV (MeOH) λmax (log ε) 256 nm (3.05), 356 nm (2.88); IR (ATR) νmax 3359, 2922,

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1662, 1593, 1497, 1411, 1337, 1312, 1217, 1117, 1093 cm-1; HR-ESI-MS (positive mode) m/z

124

515.0796 [M + Na]+ (calcd for C22H20O13Na, 515.0802); 1H- and

125

Tables 1 and 2.

13

C-NMR data are given in

126

Rhamnazin-3-O-β-D-glucuronide (2): yellowish amorphous powder; [ܽ]ଶ଴ ୈ : -1.64° (c 0.01,

127

MeOH); UV (MeOH) λmax (log ε) 254 nm (3.35), 353 nm (3.24); IR (ATR) νmax 3399, 2926,

128

1652, 1595, 1496, 1340, 1294, 1209, 1058 cm-1; HR-ESI-MS (positive mode) m/z 529.0953 [M

129

+ Na]+ (calcd for C23H22O13Na, 529.0958); 1H- and 13C-NMR data are given in Tables 1 and 2.

130

Rhamnazin-3-O-β-D-glucuronide-6′′-methyl ester (3) : yellowish amorphous powder; [ܽ]ଶ଴ ୈ

131

: -8.48° (c 0.01, MeOH); UV (MeOH) λmax (log ε) 254 nm (3.24), 354 nm (3.15); IR (ATR) νmax

132

3377, 2923, 1742, 1651, 1591, 1495, 1338, 1204, 1161, 1010 cm-1; HR-ESI-MS (positive mode)

133

m/z 543.1109 [M + Na]+ (calcd for C24H24O13Na, 543.1115); 1H- and 13C-NMR data are given in

134

Tables 1 and 2.

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Rhamnocitrin-3-O-β-D-glucuronide-6′′-methyl ester (4): yellowish amorphous powder;

136

[ܽ]ଶ଴ ୈ : -23.36° (c 0.01, MeOH); UV (MeOH) λmax (log ε) 265 nm (3.31), 347 nm (3.25); IR

137

(ATR) νmax 3323, 2951, 1746, 1658, 1597, 1500, 1443, 1414, 1345, 1287, 1213, 1181, 1085,

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1058, 1015 cm-1; HR-ESI-MS (positive mode) m/z 513.1005 [M + Na]+ (calcd for C23H22O12Na,

139

513.1009). 1H- and 13C-NMR data are given in Tables 1 and 2.

140

Acid Hydrolysis of Compounds 1 and 2. Compounds 1 and 2 (each 1 mg) was refluxed in

141

1% HCl (1 mL) for 1 h to yield aglycone and sugar. The reaction mixture was extracted with

142

EtOAc (5 mL) to give an aqueous fraction containing the sugar and EtOAc fraction containing

143

the aglycone. The aqueous fraction was concentrated and compared with authentic D-glucuronic

144

acid (Tokyo Chemical Industry, Tokyo, Japan) on silica gel TLC plates with EtOAc-MeOH-

145

H2O-AcOH (13:3:3:4), using 20% H2SO4.

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Determination of Absolute Configurations of the Sugars. The absolute configuration of

147

the glucuronic acid moiety in compounds 1 and 2 was determined by the method of Tanaka et

148

al.10 Sugars from each sample (0.5 mg) were dissolved in pyridine (0.1 mL) containing L-

149

cysteine methyl ester hydrochloride (0.5 mg) and heated at 60 °C for 1 h. o-Tolyl isothiocyanate

150

(1 µL) was then added, and the mixture was heated at 60 °C for an additional 1 h. Each reaction

151

mixture was directly analyzed by reversed-phase HPLC using a PDA-100 photodiode array

152

detector, an ASI-100 automated sample injector, and a TCC-100 Thermostatted Column

153

Compartment (Dionex, Ontario, Canada). The column used was a 250 mm × 4.6 mm i.d., 5 µm,

154

XBridge C18 5 µm (Waters, MA, USA); mobile phase MeCN−H2O (25:75, v/v) containing 50

155

mM H3PO4; detection UV (250 nm); flow rate 0.8 mL/min; column temperature 35 °C.

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Authentic methyl 2-(polyhydroxyalkyl)-3-(o-tolylthiocarbamoyl)-thiazolidine-4(R)-carboxylates

157

were prepared from

158

tolylthiocarbamoyl)-thiazolidine-4(S)-carboxylate of D-glucuronic acid was prepared by reaction

159

with D-cysteine methyl ester and o-tolylisothiocyanate since L-glucuronic acid was not available.

160

The derivatives of D-glucuronic acid in 1 and 2 were identified by comparison of their retention

161

times with those of authentic samples treated in the same way as described above (Rt:

162

glucuronic acid derivative 14.62 min, L-glucuronic acid derivative 14.01 min).

D-glucuronic

acid as described above. The enantiomeric (o-

D-

163

Antiproliferative Activity Assay. The human ovarian cancer cells (A2780) were originally

164

from American-type culture collections. Cells were cultured in RPMI supplemented with 5 %

165

fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin sulfate (100 µg/mL).

166

Cytotoxicity was assessed by MTT assays. Briefly, the cells (5 × 104) were seeded in each well

167

containing 50 µL of RPMI medium in a 96-well plate. After 24 h, cisplatin and various

168

concentrations of the compounds from cloves were added. After 48 h, 50 µL of MTT (5 mg/mL

8


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169

stock solution) were added, and the plates were incubated for an additional 4 h. The medium was

170

discarded, and the formazan blue that formed in the cells was dissolved in 50 µL of DMSO. The

171

optical density was measured at 540 nm by microplate spectrophotometer (SpectraMax;

172

Molecular Devices, Sunnyvale, CA, USA).

173 174

RESULTS AND DISSCUSSION

175

Structural Elucidation. Compound 1 was obtained as a yellowish amorphous powder, in

176

which the molecular formula was established as C22H20O13 by HR-ESI-MS (m/z 515.0796 [M +

177

Na]+ calcd for C22H20O13Na, 515.0802). The UV spectrum showed absorption bands at λmax 256

178

and 356 nm that suggested a flavonol. The 1H-NMR spectrum of 1 showed one singlet signal at

179

δH 12.54 (1H, s, 5-OH) attributed to an intramolecular hydrogen-bonded hydroxyl group. The

180

meta-coupled signals of A ring at [δH 6.71 (1H, d, J = 2.0 Hz, H-8) and 6.38 (1H, d, J = 2.0 Hz,

181

H-6)] and ABX signals of B ring at [δH 7.62 (1H, d, J = 2.0 Hz, H-2′), 7.62 (1H, dd, J = 9.0, 2.0

182

Hz, H-6′) and 6.84 (1H, d, J = 9.0 Hz, H-5′)] were observed. A sharp singlet signal was observed

183

at δH 3.86 (3H, s, 7-OCH3) for one methoxy group. Two signals in the sugar region at δH 5.50

184

(1H, d, J = 7.5 Hz) and δH 3.57 (1H, d, J = 9.5 Hz) corresponding to the anomeric proton and

185

GlcA H-5 of glucuronic acid moiety, respectively, were observed. The large coupling constant of

186

the anomeric proton signal (J = 7.5 Hz) indicated the β-configuration of the glucuronic acid

187

moiety. The

188

monoglycoside and one methoxy group in aromatic ring (δC 56.1). The most deshielded carbon

189

signal at δC 177.3 (C-4) was due to the carbonyl group in aromatic ring which was hydrogen-

190

bonded with 5-OH (δH 12.54). C-7 (δC 165.2), C-5 (δC 160.9), C-4′ (δC 148.8) and C-3′ (δC 145.0)

191

which were aromatic carbon influenced by hydroxy or methoxy substituents were shown in the

13

C-NMR spectrum of 1 displayed signals for 22 carbons including flavonol

9



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192

downfield region. The anomeric and carbonyl carbon signals of glucuronic acid moiety were

193

observed at δC 101.0 and 170.0, respectively, together with 4 carbon signals in sugar region

194

corresponding to GlcA C-2 up to GlcA C-5 (δC 71.4, 73.8, 75.9 and 75.9) indicating the existence

195

of glucuronic acid. Moreover, acid hydrolysis of 1 released D-glucuronic acid. On the HMBC

196

spectral analysis, the locations of the glucuronic acid moiety and methoxy group were assigned

197

by the long-range correlations from anomeric GlcA H-1 at δH 5.50 (1H, d, J = 7.5 Hz) to C-3 (δC

198

133.3) and from methoxy group at δH 3.86 (3H, s) to C-7 (δC 165.2) (Fig. 2). Therefore, the

199

structure of 1 was characterized as 5,3′,4′-trihydroxy-7-methoxyflavonol 3-O-β-D-glucuronide

200

(rhamnetin-3-O-β-D-glucuronide).

201

Compound 2 was isolated as a yellowish amorphous powder, in which the molecular formula

202

was established as C23H22O13 by HR-ESI-MS (m/z 529.0953 [M + Na]+; calcd for C23H22O13Na,

203

529.0958). The UV spectrum showed absorption bands at λmax 254 and 353 nm indicating 2 is

204

also a flavonol. The proton and carbon signals in the 1H- and 13C-NMR spectra of 2 were similar

205

to those of 1 except for an additional presence of one methoxy group in B ring. The 1H-NMR

206

spectrum of 2 revealed one singlet signal at δH 12.52 (1H, s, 5-OH), meta-coupled signals of A

207

ring at [δH 6.75 (1H, d, J = 2.0 Hz, H-8) and 6.39 (1H, d, J = 2.0 Hz, H-6)] and ABX system

208

signals of B ring at [δH 8.00 (1H, d, J = 2.0 Hz, H-2′), 7.54 (1H, dd, J = 8.0, 2.0 Hz, H-6′), and

209

6.92 (1H, d, J = 8.5 Hz, H-5′)]. Two aromatic methoxy group signals observed at δH 3.86 (3H, s,

210

7-OCH3) and δH 3.87 (3H, s, 3′-OCH3). Anomeric proton signal at δH 5.61 (1H, d, J = 7.0 Hz)

211

indicating the β-configuration. The

212

indicating flavonol monoglycoside with two methoxy groups. Also, the presence of a

213

glucuronic acid moiety in 2 was identified by acid hydrolysis of 2. The locations of the sugar

214

moiety and two methoxy groups were confirmed by the HMBC experiment [δH 5.61 (1H, d, J =

13

C-NMR spectrum revealed the presence of 23 carbon

10


D-

Page 11 of 26


215

7.0 Hz, GlcA H-1) / C-3 (δC 133.0), δH 3.86 (3H, s) / C-7 (δC 165.2), and δH 3.87 (3H, s) / C-3′ (δC

216

146.9)] (Fig. 2). Therefore, the structure of 2 was elucidated as 5,4′-dihydroxy-7,3′-

217

dimethoxyflavonol 3-O-β-D-glucuronide (rhamnazin-3-O-β-D-glucuronide).

218

Compound 3, a yellowish amorphous powder, had the molecular formula as C24H24O13

219

deducing from HR-ESI-MS at m/z 543.1109 [M + Na]+ (calcd for C24H24O13Na, 543.1115). The

220

UV spectrum showed strong similarities with 2 (λmax 254 and 353 nm) revealing absorption

221

bands at λmax 254 and 354 nm due to the same aglycone. Also, the NMR features of 3 closely

222

resembled those of 2 except for an additional presence of one methyl ester at GlcA C-6. The 1H-

223

NMR spectrum of 3 revealed the strong similarities with 2 showing the intramolecular hydrogen-

224

bonded hydroxyl group signal [δH 12.50 (1H, s, 5-OH)], meta-coupled signals of A ring [δH 6.74

225

(1H, d, J = 2.0 Hz, H-8) and 6.38 (1H, d, J = 2.0 Hz, H-6)], ABX signals of B ring [δH 7.96 (1H,

226

d, J = 2.0 Hz, H-2′), 7.53 (1H, dd, J = 8.5, 2.0 Hz, H-6′), and 6.93 (1H, d, J = 8.5 Hz, H-5′)], two

227

aromatic methoxy groups [δH 3.85 (3H, s, 7-OCH3), and δH 3.89 (3H, s, 3′-OCH3)], and anomeric

228

proton of glucuronic acid moiety [δH 5.62 (1H, d, J = 7.5 Hz)] which was the β-configuration.

229

However, additional singlet signal for methyl ester of glucuronic acid moiety [δH 3.58 (3H, s,

230

GlcA C-6-OCH3)] of 3 was observed in upfield region. In the 13C-NMR spectrum, all the carbon

231

signals of 3 were revealed as 24 which were similar with all those of 2 except for additional

232

carbon signal at δC 51.9 (Table 2). Through the HMBC spectral analysis, the locations of

233

glucuronic acid moiety, two methoxy groups, and one methyl ester were deduced [δH 5.62 (1H,

234

d, J = 7.5 Hz, GlcA H-1) / δC 133.0 (C-3), δH 3.89 (3H, s, OCH3) / δC 147.0 (C-3′), δH 3.85 (3H, s)

235

/ δC 165.2 (C-7), δH 3.58 (3H, s) / δC 169.1 (GlcA C-6)] (Fig. 2). Therefore, the structure of 3 was

236

determined as 5,4′-dihydroxy-7,3′-dimethoxyflavonol 3-O-β-D-glucuronide-6′′-methyl ester

237

(rhamnazin-3-O-β-D-glucuronide-6′′-methyl ester).

11



Page 12 of 26

238

Compound 4, a yellowish amorphous powder, its molecular formula C23H22O12 was

239

determined by HR-ESI-MS (m/z 513.1005 [M + Na]+; calcd for C23H22O12Na, 513.1009). The

240

UV spectrum showed maxima characteristic of λmax 265 and 347 nm indicating a flavonol. The

241

1

242

[δH 12.40 (1H, s, 5-OH)] and the meta-coupled signals of A ring at [δH 6.74 (1H, d, J = 1.5 Hz,

243

H-8) and 6.39 (1H, d, J = 2.0 Hz, H-6)] were observed. However, unlike the ABX patterns of

244

compounds 1 - 3, the 1H-NMR spectrum of 4 revealed the A2X2 spin system of B ring [δH 8.05

245

(2H, d, J = 9.0 Hz, H-2′, 6′) and 6.89 (2H, d, J = 8.5 Hz, H-3′, 5′)] due to the symmetry of H-2′ /

246

H-6′ and H-3′ / H-5′. Two sharp singlet signals were observed at δH 3.86 (3H, s, 7-OCH3) and δH

247

3.56 (3H, s, GlcA C-6-OCH3) for one methoxy group and one methyl ester, respectively. In the

248

sugar region, anomeric proton and GlcA H-5 of glucuronic acid moiety were observed at δH 5.49

249

(1H, d, J = 7.0 Hz) and δH 3.73 (1H, d, J = 10.0 Hz) indicating the β-configuration. The

250

NMR spectrum revealed the presence of 23 signals including symmetric carbons the C-2′ / C-6′

251

and C-3′ / C-5′ at δC 115.1 and δC 130.9, respectively, corresponding to the molecular formula

252

C23H22O12 deduced by HR-ESI-MS. The carbon signals at δC 101.0, 170.0 and four carbon

253

signals between 70 and 80 ppm (δC 75.5, 75.4, 73.8 and 71.5) indicated the existence of

254

glucuronic acid. The HMBC experiment exhibited the long-range correlations from anomeric

255

GlcA H-1 at δH 5.49 (1H, d, J = 7.0 Hz) to C-3 (δC 133.2) indicating the location of the sugar

256

moiety. The locations of methoxy group (C-7) and methyl ester (GlcA C-6) were also

257

determined by this experiment (Fig. 2). Comparison of the above data with those in the

258

literature11 indicated that the structure of 4 is very closely related to that of rhamnocitrin-3-O-β-

259

D-glucuronide

260

Therefore, the structure of 4 was elucidated as 5,4′-dihydroxy-7-methoxyflavonol 3-O-β-D-

H-NMR spectrum of 4 also exhibited an intramolecular hydrogen-bonded hydroxyl group signal

13

C-

except for the presence of an additional methyl ether group at GlcA C-6 in 4.

12


Page 13 of 26

261


glucuronide-6′′-methyl ester (rhamnocitrin-3-O-β-D-glucuronide-6′′-methyl ester).

262

The known compounds (5 - 19) were identified by comparing the spectroscopic data with

263

those reported in literatures to be quercetin-3-O-β-D-glucuronide (5),12 isorhamnetin-3-O-β-D-

264

glucuronide (6),13 kaempferol-3-O-β-D-glucuronide-6′′-methyl ester (7),14 quercetin-3-O-β-D-

265

glucuronide-6′′-methyl ester (8),15 isorhamnetin-3-O-β-D-glucuronide-6′′-methyl ester (9),16

266

kaempferol-3-O-β-D-glucoside (10),17 quercetin-3-O-β-D-glucoside (11),18 isorhamnetin-3-O-β-

267

D-glucoside

268

luteolin (15),22 quercetin (16),23 rhamnocitrin (17),24 kumatakenin (18),25 and pachypodol (19).26

269

Among the compounds, six flavonoids (6, 7, 9, 14, 18, and 19) were isolated from the flower

270

buds of S. aromaticum for the first time in this study.

(12),19 rhamnazin-3-O-β-D-glucoside (13),20 quercetin-7-O-β-D-glucoside (14),21

271

Antiproliferative Activities. The 70% EtOH extract of the flower buds of S. aromaticum

272

exhibited a significant inhibitory activity in a preliminary in vitro screening against human

273

ovarian cancer cells (A2780) using MTT assays (IC50 value of 22.67 µg/mL). Thus, all the

274

flavonoids (1 - 19) obtained in this study were evaluated for their antiproliferative activities

275

against human ovarian cancer cells (A2780). Some of the flavonoids showed a significant

276

cytotoxicity on A2780 cells (Table 3). Among these, pachypodol (19) exhibited the most potent

277

cytotoxicity on A2780 cells with observed IC50 value of 8.02 ± 3.23 µM comparable to cisplatin

278

(IC50 value : 6.96 ± 2.60 µM). The three flavonoid aglycones, luteolin (15), quercetin (16), and

279

kumatakenin (18), also showed significant cytotoxicity with IC50 values, 10.89, 40.39 and 19.76

280

µM, respectively, whereas rhamnocitrin (17) was not active (IC50 value > 100 µM). To our

281

knowledge, this is the first report on the antiproliferative activity of kumatakenin (18) against

282

cancer cell lines. The flavonol glucosides (10 - 12, 14) and flavonol glucuronides (1, 2, 4 - 9)

283

were not effective except for rhamnazin-3-O-β-D-glucoside (13) and rhamnazin-3-O-β-D-

13



Page 14 of 26

284

glucuronide-6″-methyl ester (3), which showed the weak cytotoxicity with IC50 values, 67.97 and

285

88.49 µM, respectively. Luteolin (15) exhibited stronger cytotoxicity than quercetin (16) which

286

has an additional hydroxyl group at C-3. Rhamnocitrin (17) which has a hydroxyl group at C-3

287

did not show cytotoxicity whereas kumatakenin (18) which has a methoxy group at C-3 was

288

cytotoxic. From these results, it was proposed that substituents at C-3 of flavone affect

289

cytotoxicity on A2780 cells. It was also observed that pachypodol (19) which has additional

290

methoxy group at C-3′ showed more potent cytotoxicity than kumatakenin (20). Thus

291

substituents at C-3′ also may influence to the cytotoxicity of flavonoids. To ensure the role of

292

substituents at C-3 and/or C-3′ of flavonoids, further studies should be performed.

293

Pachypodol (19) has been shown various biological activities in the previous studies,

294

including antiviral activity,27 antiemetic activity,28 antimutagenic activity,29 cytotoxicity against

295

KB, Hep-G2 and RD human cell lines,30 water-splitting enzyme inhibition,31 cytotoxicity on

296

Caco 2 colon cancer cell lines,32 and antitumor activity against HgpG-2, K-562, and A-549 cell

297

lines.33 However, this is the first report that pachypodol (19) possesses cytotoxicity against

298

human ovarian cancer cells (A2780). From the previous works and our study, pachypodol (19) is

299

worthy of consideration as an anti-cancer agent through additional biological evaluation.

300

In conclusion, we report here four new flavonol glucuronides and fifteen known flavonoids

301

from the flower buds of S. aromaticum (cloves) as well as their cytotoxicity on human ovarian

302

cancer cells (A2780) using MTT assays. Some of the flavonoids (3, 13, 15, 16, 18, and 19)

303

showed either significant or moderate cytotoxicity on A2780 cells. The results showed that the

304

flavonoids present in cloves attribute to its antiproliferative activity at least in parts.

305 306

ASSOCIATED CONTENT

14


Page 15 of 26

307


Supporting Information

308

The Supporting Information is available free of charge on the ACS Publications website at

309

http://pubs.acs.org.

310

NMR and HRESIMS spectra of compounds 1−4 (PDF)

311 312

AUTHOR INFORMATION

313

Corresponding Author

314

*Phone: +82 2 961 0719. Fax: +82 2 966 3885. E-mail: [email protected]

315

Funding Sources

316

This research was supported by a grant from the Bio-Synergy Research Project (NRF-

317

2015M3A9C4070483) of the Ministry of Science, ICT and Future Planning through the National

318

Research Foundation of Korea (NRF), and by Basic Science Research Program through the NRF

319

funded by the Ministry of Education (NRF-2013R1A1A2004398).

320

Notes

321

The authors declare no competing financial interest.

322 323

ACKNOWLEDGMENTS

324

We thank Korea Basic Science Institute (KBSI) for running NMR and MS experiments.

325

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326 327

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experimental animal models. Rev. Bras. Farmacogn. 2009. 19, 212−217.

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alkylphenols from clove (Syzygium aromaticum) in Salmonella typhimurium TA1535/pSK1002

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quantification of 17 bioactive constituents in Sarcandra glabra by liquid chromatography-

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electrospray ionization-mass spectrometry. Anal. Methods 2014. 6, 7989−7995.

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Kim, M. J.; Chun, W. J.; Heo, M. Y.; Kwon Y. S. Flavonoid glycosides as acetylcholinesterase

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inhibitors from the whole plants of Persicaria thunbergii. Nat. Prod. Sci. 2014. 20, 191−195.

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(14) Liu, J. -B.; Zhang, Y.; Cui, B. -S.; Li, S. Chemical constituents from twigs of Myricaria bracteata. Zhongcaoyao 2013, 44, 2661−2665.

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Jaboticabin and flavonoids from the ripened fruit of black raspberry (Rubus coreanum). Food

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current chromatography of n-butanolic fraction from Senecio giganteus (Asteraceae). Nat. Prod.

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Commun. 2009. 4, 1357−1362.

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(18) He, D.; Huang, Y.; Ayupbek, A.; Gu, D.; Yang, Y.; Aisa, H. A.; Ito, Y. Separation and

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purification of flavonoids from black currant leaves by high-speed countercurrent

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chromatography and preparative HPLC. J. Liq. Chromatogr. Relat. Technol. 2010. 33, 615−628.

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plants of Nervilia fordii (Hance) Schltr. Zhongyao Xinyao Yu Linchuang Yaoli, 2012. 23,

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Spectral assignments and reference data: 1H and

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C-NMR data of hydroxyflavone derivatives.

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Aguinaldo, A. M. Phytoconstituents from Alpinia purpurata and their in vitro inhibitory activity

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2005. 60, 627−629.

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411

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412 413

19



414

Page 20 of 26

Table 1. 1H-NMR spectral data for compounds 1-4 (DMSO-d6, 500 MHz) δH Multi (J in Hz)

Positiona 1

2

3

4

6

6.38 d (2.0)

6.39 d (2.0)

6.38 d (2.0)

6.39 d (2.0)

8

6.71 d (2.0)

6.75 d (2.0)

6.74 d (2.0)

6.74 d (1.5)

2′

7.62 d (2.0)

8.00 d (2.0)

7.96 d (2.0)

8.05 d (9.0)

3′

-

-

-

6.89 d (8.5)

5′

6.84 d (9.0)

6.92 d (8.5)

6.93 d (8.5)

6.89 d (8.5)

6′

7.62 dd (9.0. 2.0)

7.54 dd (8.0, 2.0)

7.53 dd (8.5, 2.0)

8.05 d (9.0)

GlcA-1

5.50 d (7.5)

5.61 d (7.0)

5.62 d (7.5)

5.49 d (7.0)

GlcA-2-4

3.39-3.24 m

3.33-3.25 m

3.37-3.24 m

3.37-3.25 m

GlcA-5

3.57 d (9.5)

3.61 d (9.0)

3.81 d (9.0)

3.73 d (10.0)

7-OCH3

3.86 s

3.86 s

3.85 s

3.86 s

3′-OCH3

-

3.87 s

3.89 s

-

6′′-OCH3

-

-

3.58 s

3.56 s

12.54 s

12.52 s

12.50 s

12.40 s

5-OH a

From HSQC, HMBC, and COSY results.

415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431

20


Page 21 of 26

432


Table 2. 13C-NMR spectral data for compounds 1-4 (DMSO-d6, 125 MHz)

433 δc

Positiona

a

1

2

3

4

2

156.6

156.3

156.7

156.9

3

133.3

133.0

133.0

133.2

4

177.3

177.3

177.2

177.3

5

160.9

160.9

160.9

160.8

6

98.0

98.0

98.0

98.0

7

165.2

165.2

165.2

165.2

8

92.2

92.4

92.4

92.4

9

156.2

156.6

156.3

156.3

10

104.9

104.9

105.0

104.9

1′

120.8

122.1

122.2

120.3

2′

115.2

115.2

115.2

130.9

3′

145.0

146.9

147.0

115.1

4′

148.8

149.7

149.8

160.4

5′

116.3

113.4

113.3

115.1

6′

121.7

120.6

120.6

130.9

GlcA-1

101.1

101.0

101.2

101.3

GlcA-2

73.8

74.0

74.0

73.8

GlcA-3

75.9

75.8

75.6

75.4

GlcA-4

71.4

71.6

71.7

71.5

GlcA-5

75.9

75.7

75.4

75.5

GlcA-6

169.9

170.0

169.1

168.9

7-OCH3

56.1

55.1

55.6

56.1

3′-OCH3 6′′-OCH3

-

55.6 -

56.2 51.9

51.9

From HSQC, HMBC, and COSY results.

434 435

21



436

Table 3. Cytotoxicity of flavonoids (1-19) on human ovarian cancer cells (A2780)

437 Compound

Compound

IC50 (µM) a

1

>100

11

>100

2

>100

12

>100

3

88.49 ± 8.18

13

67.97 ± 5.69

4

>100

14

5

>100

15

10.89 ± 1.21

6

>100

16

40.39 ± 6.50

7

>100

17

8

>100

18

19.76 ± 1.43

9

>100

19

8.02 ± 3.23

>100

b

6.96 ± 2.60

10 a

IC50 (µM) a

cisplatin

>100

>100

Data represent means ± SD. bCisplatin was used as assay positive control.

438 439 440 441 442 443 444 445 446 447

22


Page 22 of 26

Page 23 of 26


448

Legend to Figures

449

Figure 1. Chemical structure of compounds 1−19 isolated from the flower buds of S.

450

aromaticum.

451

Figure 2. HMBC correlations of compounds 1−4.

452 453

23



454

455 456 457 458 459

Figure 1. Byeol Ryu

24


Page 24 of 26

Page 25 of 26


460

461 462 463

Figure 2. Byeol Ryu

464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480

25



481 482 483 484 485 486 487 488

Table of Contents Graphic New Flavonol Glucuronides from the Flower Buds of Syzygium aromaticum (Clove) Byeol Ryu, Hye Mi Kim, Jin Su Lee, Chan Kyu Lee, Jurdas Sezirahiga, Jeong-Hwa Woo, Jung-Hye Choi, and Dae Sik Jang

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New Flavonol Glucuronides from the Flower Buds ... - ACS Publications

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