New Phloroglucinol Derivatives from the Fruit Tree Syzygium jambos

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New Phloroglucinol Derivatives from the Fruit Tree Syzygium jambos and Their Cytotoxic and Antioxidant Activities Guo-Qiang Li, Yu-Bo Zhang, Peng Wu, Neng-Hua Chen, ZhongNan Wu, Li Yang, Rui-Xia Qiu, Guo-Cai Wang, and Yao-Lan Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04293 • Publication Date (Web): 10 Nov 2015 Downloaded from http://pubs.acs.org on November 16, 2015

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Journal of Agricultural and Food Chemistry

New Phloroglucinol Derivatives from the Fruit Tree Syzygium jambos and Their Cytotoxic and Antioxidant Activities Guo-Qiang Li,†,§ Yu-Bo Zhang,†,§ Peng Wu,†,ǁ,§ Neng-Hua Chen,† Zhong-Nan Wu,† Li Yang,† Rui-Xia Qiu,‡ Guo-Cai Wang,*,† Yao-Lan Li*,† †

Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China

‡

Department of Food Science and Engineering, Jinan University, Guangzhou 510632, P. R. China

ǁ

International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, P. R. China

1

ACS Paragon Plus Environment


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1

ABSTRACT: Seven new phloroglucinol derivatives (1–7) were isolated from the

2

fruit tree Syzygium jambos together with four known triterpenoids (8–11) and two

3

known flavones (12–13). According to the spectroscopic analyses (IR, ESIMS,

4

HRESIMS, 1D and 2D NMR), the structures of compounds 1–7 were elucidated as

5

jambone A (1), jambone B (2), jambone C (3), jambone D (4), jambone E (5),

6

jambone F (6), and jambone G (7). All the isolates were determined for their cytotoxic

7

activities on melanoma cells by MTT assay, and compounds 10 and 11 showed potent

8

activities. Moreover, compounds 1‒2, 4‒7 and 12‒13 exhibited weak antioxidant

9

activities under FRAP and DPPH radical-scavenging assays.

10 11

KEYWORDS: Syzygium jambos, phloroglucinol derivatives, structure identification,

12

cytotoxic activity, antioxidant activity

2


Page 3 of 25


13

■ INTRODUCTION

14

The genus Syzygium (Myrtaceae) which consists of 500 species is mainly distributed

15

in tropical America and Austrilia.1 In the south of China, there are about 70 species of

16

Syzygium including Syzygium jambos (L.) Alston, and many of them are used as the

17

edible and medicinal plants. Different parts of the plants have different

18

pharmacological actions in the system of traditional medicine. The fructification is a

19

kind of common fruit in folk, and it is seen as a tonic for the brain and liver.2 The

20

barks are applied to treat asthma, bronchitis and hoarseness.2 The leaves are used as

21

the source of herbal tea in China, and their decoctions are not only used as a diuretic,

22

but also used for the treatment of rheumatism and sore eyes.2 The seeds are used to

23

treat catarrh, diarrhea, diabetes and dysentery, and the flowers can reduce fever.2

24

Syzygium jambos (Eugenia jambos) is a fruit tree which originated in Southeast Asia

25

and naturalized in India.1 Previous investigations on S. jambos had led to the isolation

26

of phenols,3,4 flavonoids5-7 and triterpenoids.8,9 The modern pharmacological studies

27

had found that the extract of S. jambos possessed anti-dermatophytic,9

28

anti-bacterial,3,10 anti-inflammatory,3,11 anti-nociceptive,1 anti-oxidant12 and liver

29

protective13 activities.

30

In our efforts to study the Chinese medicines, the chemical constituents of S.

31

jambos was investigated, leading to the isolation of seven new phloroglucinol

32

derivatives (1‒7), four known triterpenoids (8‒11) and two known flavones (12‒13).

33

These isolates were tested for their cytotoxic activities on melanoma cells by MTT 3



34

assay.

Furthermore,

their

antioxidant

35

2,2-diphenyl-1-picryhydrazyl

36

antioxidant power (FRAP) assays.

37

■ MATERIALS AND METHODS

(DPPH)

capabilities

radical-scavenging

were and

Page 4 of 25

evaluated

by

ferric-reducing

38

General Apparatus and Chemicals. A Jasco V-550 UV/VIS spectrophotometer

39

was applied for recording ultraviolet absorption spectra. IR spectroscopy were

40

scanned using a Bruker EQUINOX 55 spectrometer with KBr pellets. The 1D and 2D

41

NMR spectra were measured on a Bruker AVANCE-500 NMR spectrometer (500

42

MHz for 1H NMR; 125 MHz for 13C NMR). The ESIMS data were obtained using an

43

AB SCIEX 4000 Q-Trap mass spectrometer. HRESIMS data were determined on an

44

AB SCIEX Triple TOF 5600+ mass spectrometer. Analytical HPLC was performed

45

with a Dionex chromatograph with a P680 pump, a PDA-100 photodiode array

46

detector, and a Cosmosil C18 column (4.6 × 250 mm, 5 µm). Preparative HPLC was

47

performed on an Agilent 1100 LC series with a DAD detector using a preparative

48

Cosmosil C18 column (20 × 250 mm, 5 µm). Silica gel (200-300 mesh, Qingdao

49

Marine Chemical Inc., Qingdao, China), Sephadex LH-20 (Pharmacia Biotech,

50

Uppsala, Sweden) and reverse phase C-18 (50 µm, YMC, Kyoto, Japan) were used to

51

perform column chromatography (CC). The precoated silica gel GF254 plate for TLC

52

was purchased from Yantai Chemical Industry Research Institute (Yantai, China). All

53

reagents were provided by Tianjin Damao Chemical Company (Tianjin, China).

54

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),

55

acid,

2,4,6-tris(2-pyridyl)-s-triazine

(TPTZ)

and

L-ascorbic

2,2-diphenyl-1-picrylhydrazyl 4


Page 5 of 25


56

(DPPH) were purchased from Sigma (St. Louis, USA).

57

Plant Material. The leaves of S. jambos were collected in May 2012 from

58

Guangzhou, Guangdong, China. This plant was authenticated by Prof. Guang-Xiong

59

Zhou of the College of Pharmacy, Jinan University. A voucher specimen (NO.

60

20120524) is deposited in the Institute of Traditional Chinese Medicine and Natural

61

Products, College of Pharmacy, Jinan University (Guangzhou, China).

62

Extraction and Isolation. The air-dried and powdered leaves (10 kg) of S.

63

jambos were extracted thrice with 95% EtOH at 70°C. The solution was evaporated

64

under reduced pressure to get a residue (700 g). The residue was firstly suspended in

65

water, and then partitioned with petroleum ether, ethyl acetate, and n-butyl alcohol,

66

respectively. The ethyl acetate part (250 g) was loaded to a silica gel CC

67

(CHCl3/MeOH, 100:0 to 0:100, v/v) to give five fractions (1–5). Fraction 1 (45 g) was

68

separated by a reversed silica gel (MeOH/H2O, 30:70 to 100:0, v/v) and Sephadex

69

LH-20 (CHCl3/MeOH, 50:50, v/v) CC to obtain compounds 8 (11 mg), 9 (7 mg) and

70

11 (16 mg). Fraction 2 (3 g) was purified by Sephadex LH-20 CC (MeOH) and

71

preparative HPLC (MeOH/H2O, 65:35, v/v) to afford compounds 5 (13 mg) and 10

72

(14 mg). Fraction 3 (30 g) was separated by reversed silica gel (MeOH/H2O, 30:70 to

73

100:0, v/v) and Sephadex LH-20 (MeOH) CC to achieve compounds 1 (12 mg), 3 (10

74

mg) and 4 (22 mg). Fraction 3 was further purified by preparative HPLC (MeOH/H2O,

75

75:25, v/v) to afford compounds 2 (15 mg), 6 (8 mg) and 7 (16 mg). Fraction 5 (28 g)

76

was chromatographed twice on reversed silica gel CC (MeOH/H2O, 50:50 to 100:0,

77

v/v) and Sephadex LH-20 CC (MeOH) to yield compounds 12 (12 mg) and 13 (9 mg). 5



Page 6 of 25

78

Jambone A (1): red gum; UV (MeOH) λmax 205, 226, 286 nm; IR (KBr) νmax 3200,

79

2927, 1700, 1620, 1525, 1459, 1394, 1253, 1170, 1078, 1024, 987, 828, 733, 525

80

cm-1; 1H and

81

(calcd for C24H35O4, 387.2535).

13

C NMR data see Tables 1 and 2; HRESIMS m/z 387.2526 [M+H]+

82

Jambone B (2): red gum; UV (MeOH) λmax 204, 232, 286 nm; IR (KBr) νmax 3238,

83

2927, 2854, 1700, 1627, 1525, 1459, 1450, 1292, 1147, 1112, 723, 615 cm-1; 1H and

84

13

85

C25H35O4, 399.2535).

C NMR data see Tables 1 and 2; HRESIMS m/z 399.2530 [M-H]- (calcd for

86

Jambone C (3): red gum; UV (MeOH) λmax 204, 227, 286 nm; IR (KBr) νmax 3238,

87

3009, 2930, 2854, 1705, 1630, 1525, 1463, 1395, 1249, 1171, 1076, 1023, 725, 526

88

cm-1; 1H and

89

(calcd for C24H37O4, 389.2692).

13


90

Jambone D (4): red gum; UV (MeOH) λmax 204, 227, 286 nm; IR (KBr) νmax 3387,

91

3006, 2931, 2854, 1717, 1697, 1680, 1624, 1510, 1453, 1142, 724, 668, 446 cm-1; 1H

92

and

93

C25H37O4, 401.2692).

13

C NMR data see Tables 1 and 2; HRESIMS m/z 401.2675 [M-H]- (calcd for

94

Jambone E (5): red gum; UV (MeOH) λmax 205, 227, 249, 294 nm; IR (KBr) νmax

95

3400, 3013, 2958, 2855, 1665, 1621, 1584, 1506, 1424, 1365, 1272, 1167, 1067, 1071,

96

846, 728, 554 cm-1; 1H and

97

409.2376 [M-H]- (calcd for C26H33O4, 409.2379).

13

C NMR data see Tables 1 and 2; HRESIMS m/z

98

Jambone F (6): red gum; UV (MeOH) λmax 207, 230, 251, 297 nm; IR (KBr) νmax

99

3461, 2928, 2857, 1657, 1622, 1585, 1460, 1420, 1357, 1296, 1166, 1067, 846, 554 6


Page 7 of 25


100

cm-1; 1H and

13


101

(calcd for C27H37O4, 425.2692).

102

Jambone G (7): red gum; UV (MeOH) λmax 204, 228, 248, 294 nm; IR (KBr) νmax

103

3400, 2929, 2856, 1661, 1621, 1584, 1510, 1423, 1358, 1271, 1164, 1115, 1067, 978,

104

846, 771, 727, 551 cm-1; 1H and

105

413.2683 [M+H]+ (calcd for C26H37O4, 413.2692).

13

C NMR data see Tables 1 and 2; HRESIMS m/z

106

Cells. The melanoma SK-MEL-28 and SK-MEL-110 cells, as well as normal Vero

107

cells were provided by Sun Yat-Sen University Cancer Center and maintained in

108

DMEM medium (Gibco) containing 5% fetal bovine serum (Gibco) at 37°C in air

109

with 5% CO2.

110

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.

111

According to the early study,14 MTT assay was used to assess the cytotoxicities of

112

compounds. The cells (3000 per well) were cultivated in 96-well plates for 24 h to

113

obtain 80% confluent monolayer. The medium was then replaced with new medium

114

containing different concentrations of compounds, and the medium without

115

compounds as control. After incubation for 72 h, medium was replaced by 10 µL of

116

the MTT, and the cells were further incubated for another 4 h to allow MTT formazan

117

formation. Following incubation, the medium was replaced by DMSO (100 µL) to

118

dissolve the formazan crystals in each well. Absorbance was detected by a microplate

119

reader (Thermo Scientific, USA) at 570 nm. Each assay was performed three times,

120

and calculated the concentration giving 50% inhibition (IC50). Cisplatin was used as

121

the positive control. 7



Page 8 of 25

122

FRAP Assay. FRAP assay of compounds was estimated in triplicate according to

123

our previous report.15 10 mM TPTZ was dissolved into 40 mM HCl. The 25 mL of

124

0.3 M acetate buffer, 2.5 mL of 20 mM FeCl3 solution and 2.5 mL of 10 mM TPTZ

125

solution were mixed to prepare FRAP reagent. 20 µL of sample (100 µM) and 180 µL

126

of FRAP reagent were added to a 96-well microplate. In the darkness, the mixture was

127

shaken adequately and allowed to stand for 5 min at room temperature. And then, the

128

absorbance was detected at 593 nm with a multi-mode detection microplate reader.

129

The calibration curve was established by using different concentrations (0.15–1.5 mM)

130

of FeSO4·7H2O solution. The positive group was preformed using ascorbic acid. The

131

FRAP value of sample was expressed as the concentration (µM) generating an

132

absorbance increase equivalent to 1 mM Fe2+ solution.

133

DPPH Radical Scavenging Capacity Assay. The antioxidant activities were also

134

assessed by the scavenging activity of stable DPPH free radicals.15,16 100 µL of the

135

sample at different concentrations (0–500 µM in ethanol) was added to 100 µL of

136

DPPH solution (200 µM in ethanol) in a 96-well microplate. The mixture was shaken

137

vigorously and incubated for 30 min in the darkness. The absorbance of the mixture

138

was recorded at 517 nm by using a microplate reader, and ascorbic acid was

139

considered as the positive control. The scavenging capacity of DPPH was calculated

140

in the following way: scavenging activity (%) = 100 × (Acontrol–Asample)/Acontrol, Asample:

141

the absorbance of sample, Acontrol: the absorbance of control. Each assay was

142

determined in triplicate and calculated the concentration scavenging 50% of DPPH

143

radical (SC50). 8


Page 9 of 25


144 145

■ RESULTS AND DISCUSSION

146

The 95% ethanol extract of the leaves of S. jambos was separated by repeated silica

147

gel, ODS, Sephadex LH-20 CC and preparative HPLC to afford seven new

148

compounds, along with six known ones, which were identified as jambone A (1),

149

jambone B (2), jambone C (3), jambone D (4), jambone E (5), jambone F (6),

150

jambone

151

3β-O-trans-p-coumaroylalphitolic acid (10),19 3β-O-cis-p-coumaroylalphitolic acid

152

(11),19

153

6-desmethyl-sideroxylin (13)21 (Fig. 1).

G

(7),

oleanolic

acid

(8),17

betulinic

5,7-dihydroxy-6,8-dimethyl-4′-methoxyflavone

acid

(9),18

(12),20

154

Compound 1 was obtained as red gum. Its molecular formula, C24H34O4, was

155

established on the basis of HRESIMS (m/z 387.2526 [M+H]+, calcd for C24H35O4,

156

387.2535). The IR spectrum suggested the presence of hydroxyl (3200 cm-1), alkyl

157

(2927 cm-1), carbonyl (1700 cm-1), and aromatic ring (1620, 1525 cm-1) functionalities.

158

The 1H NMR spectrum showed a set of double-bond signals at δH 5.23-5.37 (6H,

159

overlapped), indicating the presence of three disubstituted double bonds. The four

160

characteristic proton signals at δH 2.77 (4H, overlapped) suggested the presence of

161

two doubly allylic methylenes. The 13C NMR and DEPT spectra revealed the presence

162

of 24 carbon signals, including a ketonic carbon (δC 207.3), twelve olefinic carbons

163

(δC 95.7-165.9), ten methylenes (δC 21.4-44.8) and a methyl (δC 14.6). All the data of 1

164

were

165

(Z,Z,Z,Z)-1-(2',4',6'-trihydroxyphenyl)octadeca-6,9,12,15-tetraen-l-one,22 except for

similar

to

those

of

the

known

compound

9



Page 10 of 25

166

the absence of a double bond at C-6, -7 and the presence of two methylenes at δC 30.6,

167

30.6, suggesting that the double bond at C-6, -7 was hydrogenated in 1. This was

168

confirmed by the HMBC correlations between H-8 (δH 2.03) and C-6 (δC 30.6)/C-10

169

(δC 128.7). With the aid of HSQC, 1H-1H COSY, and HMBC experiments, the 1H and

170

13

171

olefinic double bonds were indicated by the chemical shifts observed in the 13C NMR

172

spectrum for the signals of the doubly allylic methylenes (δC 26.4, C-11; δC 26.3,

173

C-14).23 Thus, the structure of 1 was elucidated and named as jambone A.

C NMR signals of 1 were assigned as shown in Table 1. The Z configurations of the

174

Compound 2 was also yielded as red gum. The molecular formula was established

175

to be C25H36O4 based on HRESIMS (m/z 399.2530 [M-H]-, calcd for C25H35O4,

176

399.2535). The IR (3238, 2927, 1700, 1627 and 1525 cm-1) and UV (204, 232 and

177

286 nm) spectra of 2 revealed that it was also a phenol derivative. The 1H and

178

NMR spectra of 2 were very similar to those of 1 except for the presence of an

179

additional methyl (δH 1.85/δC 7.3) in 2 (Tables 1 and 2). The HMBC correlations

180

between H-7′ (δH 1.85) and C-2′ (δC 163.7)/C-3′ (δC 103.5) suggested the additional

181

methyl was connected to C-3′. The 1H-13C long-range correlation of H-2 (δH

182

1.63)/C-1′ (δC 105.1) in the HMBC spectrum indicated the position of the acyl chain

183

was also at C-2′. The Z configurations of the olefinic double bonds were the same as

184

those of 1 indicated by its 13C NMR spectrum. Accordingly, compound 2 was deduced

185

and named as jambone B.

13

C

186

The HRESIMS analysis of 3 showed a quasi-molecular ion peak at m/z 389.2688

187

[M+H]+ (calcd for C25H37O4, 389.2692), corresponding to a molecular formula 10


Page 11 of 25


188

C24H36O4. Comparison of the 1H and 13C NMR data of 3 with those of 1 showed that

189

they were similar (Tables 1 and 2). The most notable difference was the double bond

190

between C-15 (δC 128.1) and C-16 (δC 132.6) was replaced by two methylenes at δC

191

30.4 (C-15) and δC 32.6 (C-16), which was verified by the HMBC correlation of H-18

192

(δH 0.87)/C-16. The structure of 3 was deduced by analysis of the HSQC, 1H-1H

193

COSY and HMBC spectra, and all the 1H and 13C NMR data of 3 were assigned and

194

shown in Tables 1 and 2. The above evidence established the structure of compound 3

195

to be jambone C.

196

The molecular formula of 4 was established as C25H38O4 based on the

197

quasi-molecular ion m/z 401.2675 [M-H]- (calcd for C25H37O4, 401.2692). The 1H, 13C

198

NMR and DEPT spectra were similar to those of 3, except for the presence of an

199

additional signal of methyl at C-3′ in 4 (Tables 1 and 2). And this was verified by the

200

HMBC correlations between H-7′ (δH 1.90) and C-2′ (δC 163.8)/C-3′ (δC 103.5). The

201

location of the acyl chain was further confirmed by the HMBC correlation between

202

H-2 (δH 3.02) and C-1′ (δC 105.1). According to the chemical shifts of the doubly

203

allylic methylene (δC 26.5, C-11), the configurations of olefinic double bonds were

204

indicated as 9Z and 12Z.23 Thus, compound 4 was identified as jambone D.

205

Compound 5 was isolated as red gum. The HRESIMS exhibited the molecular ion

206

peak at m/z 409.2376 [M-H]- (calcd for C26H33O4, 409.2379), indicating its molecular

207

formula was C26H34O4. The spectral properties of 5 [IR νmax 1665, 1621, 1584 cm−1;

208

UV (CH3OH) λmax 205, 227, 249, 294 nm] implied the presence of a

209

5,7-dihydroxychromone chromophore. Indeed, the

13

C NMR spectrum (Table 2) 11



Page 12 of 25

210

contained all the appropriate resonances (δC 95.4, 100.7, 104.6, 108.1, 160.0, 163.1,

211

168.0, 172.2, 183.7) for this ring system.24 Comparison of the 1H and 13C NMR data

212

of 5 (Table 1) with those of 1 showed that they possessed a same long side-chain. The

213

HMBC correlations between H-1′ (δH 2.55)/H-2′ (δH 1.66) and C-2 (δC 172.2)

214

revealed the side chain was located at C-2. The

215

methylenes (δC 26.5, C-10′; δC 26.4, C-13′) revealed the olefinic double bonds were Z

216

configurations.23 Accordingly, compound 5 was deduced to be jambone E.

13

C NMR data of the doubly allylic

217

The 1D NMR data of compound 6 were in good agreement with those of

218

compound 5 (Tables 1 and 2), except for the presence of an additional methyl (δH

219

1.97/δC 7.3) in 6. The HMBC correlations between H-11 (δH 1.97) and C-5 (δC

220

160.0)/C-6 (108.8) suggested the additional methyl was connected to C-6. The

221

location of the alkyl chain was further confirmed by the HMBC correlation between

222

H-2′ (δH 1.65) and C-2 (δC 172.1). The configurations of the olefinic double bonds

223

were also established by its

224

deduced to be jambone F.

13

C NMR spectrum.23 Accordingly, compound 6 was

225

Compound 7 was also isolated as red gum. Its molecular formula was determined

226

as C26H36O4 according to the HRESIMS (m/z 413.2683 [M+H]+, calcd for C26H37O4,

227

413.2692). The 1H and 13C NMR data of 7 were similar to those of 5 (Tables 1 and 2).

228

The most notable difference was that a double bond at C-14′, 15′ (δC 128.2, 132.7) in

229

5 was replaced by two methylenes (δC 30.4, C-14′; δC 32.6, C-15′) in 7, which was

230

confirmed by the HMBC correlation of H-17′ (δH 0.87)/C-15′. The structure of 7 was

231

confirmed by analysis of the HSQC, 1H-1H COSY and HMBC spectra, and all the 1H 12


Page 13 of 25


232

and 13C NMR data of 7 were assigned and shown in Tables 1 and 2. So compound 7

233

was identified as jambone G.

234

The cytotoxic activities on melanoma cells of the isolates. All the isolates were

235

tested for their cytotoxic effects on melanoma SK-MEL-28 and SK-MEL-110 cells,

236

as well as normal Vero cells using the MTT assay. As shown in Table 3, compound

237

1 possessed moderate to weak activity against SK-MEL-110 cells, and compounds 3,

238

8 and 9 exhibited moderate inhibitory effects on melanoma SK-MEL-28 and

239

SK-MEL-110 cells. It is noteworthy that compounds 10 and 11, lupane-type

240

triterpenes with coumaroyl moiety at the C-3 position, displayed more potent effects

241

on these two melanoma cells. Our result was consistent with a previous study which

242

indicated that coumaroyl moiety significantly influenced the cytotoxicity of

243

lupane-type triterpene.25

244

The antioxidant activities of the isolates. In a previous study, the extract of

245

leaves of S. jambos showed potent antioxidant capacity, which was mainly caused by

246

gallic and chlorogenic acids, rutin, quercetin, caffeic acid as well as kaempferol.12 In

247

our work, the antioxidant activities of compounds 1‒13 were also tested by FRAP and

248

DPPH radical scavenging capacity assays, however only 1‒2, 4‒7 and 12‒13

249

exhibited weak antioxidant activities (Table 4), suggesting these thirteen compounds

250

were not the major antioxidant constituents of the plant.

13



Page 14 of 25

■ ASSOCIATED CONTENT Supporting Information

The HRESIMS and NMR spectra of compounds 1–7. This material is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION Corresponding Author * (G. C. Wang) E-mail: [email protected]. * (Y.L. Li) E-mail: [email protected]. Author Contributions §

These authors contributed equally to this study.

Funding This work was supported financially by the Natural Science Foundations of China (81202429, 81273390 and 81473116), the Fundamental Research Funds for the Central Universities (11615305), the Natural Science Foundation of Guangdong Province (No. S2013020012864) and 111 Project (NO. B13038). Notes The authors declare no competing financial interest.

14


Page 15 of 25


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Warrier, P. K.; Nambiar, V. P.; Ramankutty, C. Indian medicinal plants. A

compendium of 500 species. Chennai: Orient Longman Ltd 1996, 5, 229–231. (3)

Sharma, R.; Kishore, N.; Hussein, A.; Lall, N. Antibacterial and

anti-inflammatory effects of Syzygium jambos L. (Alston) and isolated compounds on acne vulgaris. BMC Complementary Altern. Med. 2013, 13, 292. (4)

Simões-Pires, C. A.; Vargas, S.; Marston, A.; Ioset, J. R.; Paulo, M. Q.;

Matheeussen, A.; Maes, L. Ellagic acid derivatives from Syzygium cumini stem bark: investigation of their antiplasmodial activity. Nat. Prod. Commun. 2009, 4, 1371–1376. (5)

Sharma, B.; Viswanath, G.; Salunke, R.; Roy, P. Effects of flavonoid-rich

extract from seeds of Eugenia jambolana (L.) on carbohydrate and lipid metabolism in diabetic mice. Food Chem. 2008, 110, 697–705. (6)

Jayasinghe, U. L.; Ratnayake, R. M.; Medawala, M. M.; Fujimoto, Y.

Dihydrochalcones with radical scavenging properties from the leaves of Syzygium jambos. Nat. Prod. Res. 2007, 21, 551–554. (7)

Slowing, K.; Söllhuber, M.; Carretero, E.; Villar, A. Flavonoid glycosides

from Eugenia jambos. Phytochemistry 1994, 37, 255–258. (8)

Gupta, G. S.; Sharma, D. P. Triterpenoid and other constituents of Eugenia

jambolana leaves. Phytochemistry 1974, 13, 2013–2014.

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Kuiate, J. R.; Mouokeu, S.; Wabo, H. K.; Tane, P. Antidermatophytic

triterpenoids from Syzygium jambos (L.) Alston (Myrtaceae). Phytother. Res. 2007, 21, 149–152. (10) Djipa, C. D., Delmee, M.; Quetin-Leclercq, J. Antimicrobial activity of bark extracts of Syzygium jambos (L.) alston (Myrtaceae). J. Ethnopharmacol. 2000, 71, 307–313. (11) Slowing, K.; Carretero, E.; Villar, A. Anti-inflammatory activity of leaf extracts of Eugenia jambos in rats. J. Ethnopharmacol. 1994, 43, 9–11. (12) Bonfanti, G., Bitencourt, P. R.; Bona, K. S.; Silva, P. S.; Jantsch, L. B.; Pigatto, A. S.; Boligon, A.; Athayde, M. L.; Gonçalves, T. L.; Moretto, M. B. Syzygium jambos and Solanum guaraniticum show similar antioxidant properties but induce different enzymatic activities in the brain of rats. Molecules 2013, 18, 9179–9194. (13) Islam, M. R.; Parvin, M. S.; Islam, M. E. Antioxidant and hepatoprotective activity of an ethanol extract of Syzygium jambos (L.) leaves. Drug Discov. Ther. 2012, 6, 205–211. (14) Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. (15) Zhang, X. L.; Guo, Y. S.; Wang, C. H.; Li, G. Q.; Xu, J. J.; Chung, H. Y.; Ye, W. C.; Li, Y. L.; Wang, G. C. Phenolic compounds from Origanum vulgare and their antioxidant and antiviral activities. Food Chem. 2014, 152, 300–306. 16


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(16) Xu, J. J.; Wu, X.; Li, M. M.; Li, G. Q.; Yang, Y. T.; Luo, H. J.; Huang, W. H.; Chung, H. Y.; Ye, W. C.; Wang, G. C.; Li, Y. L. Antiviral activity of polymethoxylated flavones from “Guangchenpi”, the edible and medicinal pericarps of Citrus reticulata ‘Chachi’. J. Agric. Food Chem. 2014, 62, 2182–2189. (17) Guo, F.; Lin, S.; Li, Y. Isolation and identification of triterpenoids from Schefflera arboricola. Chin. J. Med. Chem. 2005, 15, 294–296. (18) Ma, Z. Z.; Hano, Y.; Nomura, T.; Chen, Y. J. Three new triterpenoids from Peganum nigellastrum. J. Nat. Prod. 2000, 63, 390–392. (19) Yagi, A.; Koda, A.; Inagaki, N.; Haraguchi, Y.; Noda, K.; Okamura, N.; Nishioka, I. Studies on the constituents of Zizyphi fructus. I. Structure of three new para-coumarates of alphitolic acid. Chem. Pharm. Bull. 1978, 26, 1798–1802. (20) Nazreen, S.; Kaur, G.; Alam, M. M.; Shafi, S.; Hamid, H.; Ali, M.; Alam, M. S. New flavones with antidiabetic activity from Callistemon lanceolatus DC. Fitoterapia 2012, 83, 1623–1627. (21) Tu, P. F.; Tao, J.; Hu, Y. Q.; Zhao, M. B. Flavones from the wood Dracaena cochinchinensis. Chin. J. Nat. Med. 2003, 1, 27–29. (22) Kazlauskas, R.; King, L.; Murphy, P. T.; Warren, R. G.; Wells, R. J. New metabolites from the brown algal genus Cystophora. Aust. J. Chem. 1981, 34, 439–447. (23) Tringali, C.; Piattelli, M. Two chromone derivatives from the brown alga Zonaria tournefortii. Tetrahedron Lett. 1982, 23, 1509–1512. (24) Buske, A.; Schmidt, J.; Porzel, A.; Adam, G. Benzopyranones and ferulic 17



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acid derivatives from Antidesma membranaceum. Phytochemistry 1997, 46, 1385–1388. (25)

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triterpenoids from the fruits of Zizyphus jujuba. Planta Med. 2003, 69, 1051–1054.

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Page 19 of 25


OH O R

1'

3'

HO

3

5

7

9

1

11

R HO

OH O R

17

OH O

1R=H 2 R = CH3 5

14

OH

5'

15

12

OH

4

10

3R=H 4 R = CH3

2

HO 7

9 O

1'

5R=H 6 R = CH3

OH O

HO

O 7

O

O R1

OH HO

OH

R2 9 R1 = H R2 = OH 10 R1 = OH R2 = trans-p-counaroyl 11 R1 = OH R2 = cis-p-counaroyl

8

O HO

O

OH O

OH O

O

OH O

12

13

Fig. 1 Structures of compounds 1–13

19



Page 20 of 25

Fig.2 Key 1H-1H COSY and HMBC correlations of 1 and 5

20


Page 21 of 25


Table 1. 1H and 13C NMR data for 1–4 (500 and 125 MHz, CD3OD, J in Hz)a No.

1 δH 3.01 t (7.3) 1.63 m 1.22-1.40 1.22-1.40 1.22-1.40 1.22-1.40 2.03 5.23-5.37 5.23-5.37 2.77 5.23-5.37 5.23-5.37 2.77 5.23-5.37 5.23-5.37 2.04 0.93 t (7.5)

2 δC 207.3 44.8 26.1 30.2b 30.4b 30.6b 30.6b 28.1 131.0 128.7 c 26.4 129.1 129.1 26.3 128.1 c 132.6 21.4 14.6 105.3 165.7 95.7 165.9 95.7 165.7

δH 2.96 t (7.5) 1.63 m 1.23-1.33 1.23-1.33 1.23-1.33 1.23-1.33 2.00 5.15-5.38 5.15-5.38 2.74 5.15-5.38 5.15-5.38 2.74 5.15-5.38 5.15-5.38 2.01 0.89 t (7.5)

δC 1 207.5 2 44.9 3 26.3 4 30.2 5 30.4b 6 30.6b 7 30.7b 8 28.1 9 131.1 128.8c 10 11 26.5 12 129.1d 13 129.2d 26.4 14 128.2c 15 16 132.7 17 21.4 14.6 18 105.1 1′ 163.7 2′ 5.80 d (2.0) 103.5 3′ 164.9 4′ 5.80 d (2.0) 5.83 s 94.8 5′ 161.3 6′ 1.85 s 7.3 7′ a Overlapped signals were reported without designating multiplicity. b-d Assignments may be intermixed.

3 δH 3.01 t (7.8) 1.63 m 1.23-1.40 1.23-1.40 1.23-1.40 1.23-1.40 2.03 5.25-5.40 5.25-5.40 2.75 5.25-5.40 5.25-5.40 2.04 1.23-1.40 1.28 1.26 0.87 t (7.5)

5.79 d (2.0) 5.79 d (2.0) -

4 δC 207.3 44.8 26.1 30.2b 30.4b 30.6b 30.7b 28.1 130.8c 129.0 26.5 129.0 130.8c 28.1 30.4 32.6 23.6 14.4 105.3 165.8 95.7 166.0 95.7 165.8

δH 3.02 t (7.5) 1.65 m 1.23-1.40 1.23-1.40 1.23-1.40 1.23-1.40 2.03 5.25-5.40 5.25-5.40 2.76 5.25-5.40 5.25-5.40 2.04 1.23-1.40 1.28 1.26 0.88 t (7.5)

5.89 s 1.90 s

21


δC 207.5 44.9 26.3 30.2b 30.4b 30.6b 30.7b 28.1 130.9 129.0 26.5 129.0 130.9 28.1 30.4 32.6 23.6 14.4 105.1 163.8 103.5 164.9 94.8 161.3 7.3


Page 22 of 25

Table 2. 1H and 13C NMR data for 5–7 (500 and 125 MHz, CD3OD, J in Hz)a No.

a

5

6

7

2

δH -

δC 172.2

δH -

δC 172.1

δH -

δC 172.3

3

5.95 s

108.1

5.97 s

108.2

5.99 s

108.3

4

-

183.7

-

184.0

-

183.8

5

-

163.1

-

160.0

-

163.2

6

6.21 d (2.0)

100.7

-

108.8

6.15 s

100.0

7

-

168.0

-

163.8

-

165.8

8

6.09 d (2.0)

9

-

160.0

-

157.5

-

159.7

10

-

104.6

-

104.8

-

105.2

11

-

-

7.3

-

-

1′

2.55 t (7.6)

34.9

2.55 t (7.5)

34.9

2.57 t (7.3)

34.9

2′

1.66 m

27.8

1.65 m

27.8

1.68 m

27.8

95.4

b

6.30 s

1.97 s

93.8

6.27 s

94.9

1.24-1.38

29.9

b

1.22-1.40

30.0b

3′

1.22-1.38

30.0

4′

1.22-1.38

30.1b

1.24-1.38

30.0b

1.22-1.40

30.1b

5′

1.22-1.38

30.1b

1.24-1.38

30.0b

1.22-1.40

30.2b

6′

1.22-1.38

30.6b

1.24-1.38

30.5b

1.22-1.40

30.6b

7′

2.02

8′

5.22-5.35

131.0c

5.22-5.35

131.0

5.24-5.37

130.8c

9′

5.22-5.35

128.8c

5.22-5.35

128.2c

5.24-5.37

129.0d

10′

2.75

11′

5.22-5.35

129.1d

5.22-5.35

129.1

5.24-5.37

129.1d

12′

5.22-5.35

129.2d

5.22-5.35

129.1

5.24-5.37

130.9c

13′

2.75

14′

5.22-5.35

128.2c

5.22-5.35

128.8c

15′

5.22-5.35

132.7

5.22-5.35

16′

2.01

21.4

17′

0.89 t (7.5)

14.6

28.1

26.5

26.4

2.02

2.73

2.73

28.0

26.5

26.3

2.02

28.1

2.74 t (6.2)

26.5

2.01

28.1

1.22-1.40

30.4

132.7

1.26

32.6

2.01

21.4

1.25

23.6

0.89 t (7.5)

14.6

0.87 t (6.8)

14.4

Overlapped signals were reported without designating multiplicity. Assignments may be intermixed.

b-d

22


Page 23 of 25


Table 3. Cytotoxic activities of the active compounds a IC50 (µM) b

Compounds SK-MEL-28 1 3 8 9 10 11 Cisplatin

c

>100 39.6 ± 0.6 30.8 ± 1.5 53.1 ± 2.3 19.3 ± 0.9 27.5 ± 1.0 11.5 ± 0.5

SK-MEL-110

Vero

75.6 ± 3.6 50.4 ± 1.3 31.5 ± 2.1 43.5 ± 1.7 18.3 ± 0.4 25.9 ± 0.5 26.5 ± 1.3

82.9 ± 6.7 63.7 ± 5.2 >100 >100 26.2 ± 2.1 81.5 ± 5.9 11.3 ± 0.4

a

Data represented as the mean value ± SD. The test concentrations ranged from 0 to 100 µM, and the IC50 value was tested by MTT assay after incubation for 72 hours. c The IC50 value of sample was higher than 100 µM.

b

23



Page 24 of 25

Table 4. Antioxidant activities of compounds 1–13 a Compounds

DPPH SC50

(µM) b

411.8 ± 15.7 1 377.9 ± 8.3 2 d 3 >500 4 487.0 ± 5.7 350.4 ± 6.3 5 316.0 ± 2.1 6 387.1 ± 13.6 7 >500 8 9 >500 >500 10 >500 11 427.2 ± 12.7 12 439.6 ± 8.0 13 Ascorbic acid 18.2 ± 0.4 a Data are represented as mean ± SD. b The test concentrations ranged from 0 to 200 µM. c The test concentrations was 100 µM. d The SC50 value of sample is higher than 500 µM. e n.d. Not detectable.

FRAP value (µM) c 30.1 ± 0.7 38.8 ± 4.9 n.d. e n.d. 43.4 ± 3.6 50.5 ± 1.5 29.2 ± 0.7 n.d. n.d. n.d. n.d. 36.6 ± 3.1 28.3 ± 4.4 457.8 ± 5.8

24


Page 25 of 25


Graphic for table of contents

25


New Phloroglucinol Derivatives from the Fruit Tree Syzygium jambos

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