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Three Novel Alkaloids from Portulaca oleracea L. and Their Anti-inflammatory Effects Cuiyu Li, Yihan Meng, Zheming Ying, Nan Xu, Dong Hao, Mingzhe Gao, Wenjie Zhang, Liang Xu, Yucong Gao, and Xixiang Ying J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02673 • Publication Date (Web): 09 Jul 2016 Downloaded from http://pubs.acs.org on July 9, 2016

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Journal of Agricultural and Food Chemistry

Three Novel Alkaloids from Portulaca oleracea L. and Their Anti-inflammatory Effects Cui-Yu Li†#, Yi-Han Meng†#, Zhe-Ming Ying‡, Nan Xu†, Dong Hao†, Ming-Zhe Gao§, Wen-Jie Zhang†, Liang Xu†, Yu-Cong Gao†, Xi-Xiang Ying†* †

School of Pharmacy, Liaoning University of Traditional Chinese Medicine, 116600,

Dalian, China ‡

School of The First Clinic, Liaoning University of Traditional Chinese Medicine,

110032, Shenyang, China §

Dalian Institute of Chemical Physics, 116023, Dalian, China

*Tel: +86 159 9854 1928; Fax: +86 0411-85890128 : E-mail: [email protected].

1

ACS Paragon Plus Environment


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1

ABSTRACT

2

Three novel carbon skeleton alkaloids named oleracimine, 1, oleracimine A, 2, and

3

oleracone A, 3, with one novel azulene carbon skeleton compound, oleracone B, 4,

4

and one known compound, β-carboline, 5, were first isolated from Portulaca oleracea

5

L. The structures were determined using spectroscopic methods including 1D and 2D

6

NMR, and HR-ESI-TOF-MS techniques. In addition, oleracimine, 1, was used to

7

investigate the anti-inflammatory effects on lipopolysaccharide (LPS) stimulated

8

macrophages; the results of ELISA, western blot and real-time PCR showed that

9

oleracimine,

1,

remarkably

inhibited

nitric

oxide

production

and

could

10

dose-dependently decrease the secretions of IL-6, TNF-α, NO and PGE2 in cell

11

culture supernatants as well as the mRNA of COX-2 and iNOS.

12

KEYWORDS: Portulaca oleracea L.; alkaloids; anti-inflammatory effects

13 14 15 16 17 18 19 20 21 22 2


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23

INTRODUCTION

24

Portulaca oleracea L., taxonomically belonging to the family of Portulacaceae, is

25

widely distributed in temperate and tropical areas. 1 P. oleracea is a common plant and

26

is distributed throughout the world. Additionally, P. oleracea is an important

27

vegetable crop rich in essential omega-3 and 6 fatty acids, α-linolenic acid,

28

α-tocopherol, β-carotene, ascorbic acid, glutathione2 and dietary minerals such as K,

29

Mg, P, Ca and Fe. 3 The plant are edible with salty taste and a slightly acidic, which is

30

often as a potherb added to soups and salads in the Mediterranean region and in

31

tropical Asian countries.

32

seeds of this plant into flour for use in mush and bread.

33

phytochemicals in vegetables and fruits provides overlapping or complementary

34

effects that contribute to a health protective effect.

35

important role as a new cultivated vegetable to address the lack of vegetable sources

36

of ω-3 fatty acids, inadequate cultivable land and escalating salinity. 7, 8

4

Native Americans and aborigines of Australia ground the

6

5

The complex mixture of

Therefore, P. oleracea plays an

37

In China, P. oleracea is regarded as the plant of “the same source of food and

38

medicine”. As a food, the plant was known as the “vegetable for long life” in China

39

because of its safety for daily consumption as a vegetable 9 and because of its many

40

health functions, i.e., its anti-oxidant,

41

analgesiac,

42

corresponding bioactive constituents have been insufficient. Many compounds have

43

been isolated from P. oleracea, including terpenes,

44

flavonoids

12

17

neuroprotective,

and alkaloids.

13

10

antibacterial,

11

and anti-aging effects.

18-20

15

14

anti-inflammatory and Studies regarding the

phenolic acids, coumarins,

16

Some of the alkaloids presented diverse 3



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45

pharmacological properties, and for example, oleracone, that was isolated from P.

46

oleracea in our laboratory, presented remarkable anti-inflammatory effects with high

47

bioavailability in rats. 21

48

In this study, we isolated for the first time three alkaloids with a novel skeleton, one

49

novel azulene carbon skeleton compound, and one known compound, from P.

50

oleracea. Their structures were determined using spectroscopic methods including 1D

51

NMR, 2D NMR, and HR-ESI-TOF-MS. Considering the healthcare functions, the

52

folkloric uses of P. oleracea and the anti-inflammatory effect of the alkaloids

53

compound 1, the most abundant among the five, was chosen as the candidate for

54

investigation of an anti-inflammatory effect on the experimental models of

55

lipopolysaccharide (LPS)-induced RAW 264.7 cells using an assay of inflammatory

56

mediators in the culture media and the protein and mRNA expression of COX-2 and

57

iNOS.

58

MATERIALS AND METHODS

59

Instruments

60

IR200 spectrophotometer (Thermo Electron Corporation, Waltham, MA). U-3010

61

spectrophotometer (Hitachi Ltd, Tokyo, Japan). AVANCE 500 MHz instrument

62

(Bruker Corporation, Switzerland). Autopol I automatic polarmeter (Rudolph

63

Research analytical, Hackettstown, NJ). 6520 quadrupole-time of flight mass

64

spectrometer (Agilent, Palo Alto, CA). Thermal Values Analyzer with Microscope

65

(Jingke, Shanghai, China). A Nexera X2 UHPLC LC-30A system (Shimadzu, Kyoto,

66

Japan). 4


21

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67

Plant Materials and Reagents

68

The air-dried aerial segments of P. oleracea were from Shijiazhuang, Hebei, China in

69

June 2014 and were identified by Xixiang Ying. A voucher specimen (No. 20140312)

70

was deposited in our laboratory. ODS and Sephadex LH-20 were purchased from GE

71

Healthcare (Marlborough, MA). Methanol and acetonitrile were HPLC grade (Damao

72

Chemical Reagent Co.,Tianjin, China). The purified water was from a Milli-Q water

73

purification system (Millipore, Bedford, MA). Other reagents of analytical grade were

74

from Jinfeng Chemical Co. (Tianjin, China). RAW 264.7 cells (TIB-71) were from the

75

American Type Culture Collection (Rockefeller, MD). Dulbeccoʹs modified Eagleʹs

76

medium (DMEM), a penicillin-streptomycin solution and fetal bovine serum (FBS)

77

(Hyclone, Logan, UT). LPS (Escherichia coli strain 0111:B4) and dimethyl sulfoxide

78

(DMSO) (Sigma-Aldrich, Santa Clara, CA). Griess reagent (Beyotime Biotechnology,

79

Shanghai, China). PGE2 enzyme-linked immunosorbent assay (ELISA) kits (Cayman,

80

Ann Arbor, MI).

81

Extraction and Isolation

82

The dried aerial segments of P. oleracea (150 kg) were crushed then extracted twice

83

with a 10-fold amount of water for 2 h each time. The combined aqueous extract was

84

condensed and then partitioned with a 2-fold amount of ethyl acetate three times to

85

provide the dried extract (200 g) then subjected to chromatography on a 200-300

86

mesh silica-gel column (120 cm × 8 cm, diam., approx. 2.5 kg), eluented gradiently

87

with petroleum and acetone (1:1, 1:2, 1:3, 1:5, v/v) to obtain 150 fractions (400 mL

88

each). After spotting the fractions on a TLC plate and spraying it with Dragendorff 5



89

reagent, fractions of 90-130 turned red-brown were combined and repeatedly

90

subjected to chromatography on a 20-40 µm ODS column (25 mc x 3 cm diam.,

91

approx. 150 g, Ultimate XB-C18) and eluted with methanol and water (50:50, v/v) to

92

obtain 10 fractions (200 mL each). The fraction 3 was purified with a Sephadex

93

LH-20 column (150 cm × 2 cm diam., approx. 100 g) and eluted with methanol-water

94

(70:30, v/v) to obtain compound 1 (30 mg, purity > 98% with UHPLC) and

95

compound 2 (8 mg, purity > 97% with UHPLC). The fraction 5 was purified with a

96

Sephadex LH-20 column (150 cm × 2 cm diam., approx. 100 g) and eluted with

97

methanol-water (60:40, v/v) to obtain compound 3 (10 mg, purity > 97% with

98

UHPLC). The fraction 6 was purified with a Sephadex LH-20 column (150 cm × 2 cm

99

diam., approx. 100 g) and eluted with methanol-water (70:30, v/v) to obtain

100

compound 4 (7 mg, purity > 97% with UHPLC). The fraction 8 was purified with a

101

Sephadex LH-20 column (150 cm × 2 cm diam., approx. 100 g) and eluted with

102

methanol-water (70:30, v/v) to obtain compound 5 (15 mg, purity > 98% with

103

UHPLC). The compounds were fully characterized by HR-ESI-TOF-MS, 1H-NMR

104

and 13C-NMR spectroscopy (Table 1). Oleracimine, 1: yellow amorphous powder; [α]D 20 +15 (c 0.1, MeOH); IR (KBr)

105 106

νmax 3354, 2958, 1666, 1554, 1510, and 1049 cm-1; UV (MeOH) λmax: 448 and 272 nm;

107

1

108

287.2118 [M + H]+ (calcd: C18H27N2O, 287.2261).

H and

13

C NMR spectroscopic data (CDCl3), see Table 1; HR-ESI-TOF-MS m/z

109

Oleracimine A, 2: yellow amorphous powder; [α]D 20 -13 (c 0.1, MeOH); IR (KBr)

110

νmax 3470, 3290, 1712, 1668, 1628, 1578, 1531, and 1312 cm-1; UV (MeOH) λmax: 446 6


Page 6 of 37

Page 7 of 37


1

H and

13

111

and 268 nm;

C NMR spectroscopic data (CDCl3), see Table 1;

112

HR-ESI-TOF-MS m/z 301.1912 [M + H]+ (calcd: C18H25N2O2, 301.2097).

113

Oleracone A, 3: brown crystal; [α]D 20 +20 (c 0.1, MeOH); IR (KBr) νmax 3330,

114

3278, 3075, 1655, 1620, 1506, 1258 and 1160 cm-1; UV (MeOH) λmax:339 and 273

115

nm; 1H and 13C NMR spectroscopic data (CDCl3), see Table 1; HR-ESI-TOF-MS m/z

116

305.2359 [M + H]+ (calcd: C18H29N2O2, 305.2413).

117

Oleracone B, 4: colorless oil; [α]D 20 0 (c 0.1, MeOH); IR (KBr) νmax 3420, 1760,

118

1659, 1625, 1310, 1205, and 893 cm-1; UV (MeOH) λmax: 295 nm; 1H and 13C NMR

119

spectroscopic data (CDCl3), see Table 1; HR-ESI-TOF-MS m/z 207.1389 [M + H]+

120

(calcd: C13H19O2, 207.1318).

121

Cell Culture

122

The macrophage cell line RAW 264.7 was maintained in DMEM supplemented with

123

10% heat-inactivated fetal bovine serum and antibiotics (100 U/mL penicillin and 100

124

µg/mL streptomycin) and incubated at 37 ºC in a humidified incubator with 5% CO2.

125

Cell Viability

126

The

127

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

128

RAW 264.7 cells were plated at an initial density of 1×104/well in 96-well plates.

129

After a 24-h incubation, the cells were pre-treated with or without various

130

concentrations (1, 4, 10, 20, or 50 µM) of oleracimine, 1, for 1 h, followed by 1

131

µg/mL LPS for 24 h; the media was then removed, and the cells were incubated with

132

5 mg/mL MTT solution for 4 h at 37 ºC. The formazan was dissolved in 150 µL

cytotoxicity

of

oleracimine,

1,

7


was

assessed

by


133

dimethylsulfoxide (DMSO), and the absorbance was detected at 570 nm by a

134

microplate reader; the untreated group was considered as 100% viable.

135

Nitric Oxide Analysis

136

The concentration of NO in the medium was measured using Griess reagent.

137

Dexamethasone (Dex) was used as a positive control. RAW 264.7 cells at a density of

138

2 × 105 were seeded in 24-well plates for 24 h and incubated with or without the

139

indicated concentrations (1, 4, 10, or 20 µM) of oleracimine, 1, for another 1 h and

140

then challenged with LPS (1 µg/mL) for 24 h. A 100-µL sample of the cell culture

141

supernatant in different concentrations was mixed with 100-µL Griess reagent. The

142

production of NO was measured at 550 nm and was compared with a sodium nitrite

143

standard calibration curve.

144

Determination of IL-6, PGE2 and TNF-α production

145

Supernatants of LPS-induced RAW 264.7 cells pre-incubated with or without the

146

indicated concentrations of oleracimine, 1, were collected and applied to quantitate

147

the production of IL-6, PGE2 and TNF-α using enzyme-linked immunosorbent assay

148

(ELISA) kits (Cayman Chemical, Ann Arbor, MI). NS-398 was used as the positive

149

PGE2 release inhibitor, and Dex was used for IL-6 and TNF-α.

150

Western Blot Analysis

151

After the indicated treatment, the cells were harvested then rinsed twice with ice-cold

152

phosphate-buffered saline (PBS) and lysed immediately by sonication with RIPA lysis

153

buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) on ice to detect

154

β-actin, COX-2 and iNOS. The concentrations of proteins were determined by BCA 8


Page 8 of 37

Page 9 of 37


155

assays (Sangon-Bio, Shanghai, China) following the manufacturerʹs protocol. For

156

Western blot analysis, thirty micrograms of total protein were subjected to 10%

157

SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes

158

(Bio-Rad, Hercules, CA). Then, the PVDF membranes were blocked in 5% non-fat

159

milk at room temperature for 2 h. After several washes in Tris-buffered saline with

160

Tween 20 (TBST), the membranes were incubated at 4 ºC overnight in diluted

161

primary antibodies (Cell Signaling Technology, Danvers, MA). The membranes were

162

then washed and hybridized with the corresponding HRP-conjugated secondary

163

antibodies (ZSGB-Bio, Beijing, China) at 37 ºC for 45 min. The blots were washed

164

three times, developed using enhanced chemiluminescence substrate (Thermo,

165

Rockford, IL) and exposed to capture the images. The density of immunoreactive

166

bands was visualized by Tanon-5200 (Tanon, Shanghai, China) and normalized to

167

β-actin.

168

Real-time PCR Analysis

169

To perform real-time PCR, RNA extraction was conducted according to the

170

manufacturerʹs instructions for the Eastep Total RNA Extraction Kit (Promega,

171

Beijing, China); a Nanodrop2000c Spectrophotometer (Thermo Scientific, Waltham,

172

MA) was used to assess the qualitative and quantative of the total RNA. In brief,

173

cDNA synthesis was conducted using the GoScript Reverse Transcription System kit

174

(Promega, Beijing, China). Total cellular RNA (5 µg) of different samples were mixed

175

with oligo (dT) primer and brought to a final volume of 5 µL with nuclease-free water.

176

The RNA and primer mixtures were heated for 5 min at 70 ºC and then immediately 9



Page 10 of 37

177

cooled on ice until the reverse transcription mixtures containing dNTP, reverse

178

transcriptase, 5 × reaction buffer, RNase inhibitor and reverse transcriptase were

179

prepared. A total of 15 µL of the reverse transcription mix and 5 µL of total cellular

180

RNA and primer mixtures were incubated at 25 ºC for 5 min, followed by heating at

181

42 ºC for 1 h and inactivation at 70 ºC for 10 min. The prepared cDNA was stored at

182

-20 ºC until analysis. The reactions were carried out in a volume of 20 µL in

183

commercial kit (TaKaRa-Bio, Dalian, China) using the following cycle parameters: 95

184

ºC, 30 s; 40 cycles, 95 ºC, 5 s, 60 ºC, 31 s. The house-keeping gene β-actin was

185

considered as the internal control, and amplifications were performed using an

186

Applied Biosystems real-time PCR detection system. The sequences of the primers

187

were as follows: iNOS (forward 5ʹ-GGTGAAGGGACTGAGCTGTT-3ʹ and reverse

188

5ʹ-ACGTTCTCCGTTCTCTTGCAG-3ʹ),

COX-2

(forward

189

5ʹ-TGGTGCCCTGGTCTGATGATG-3ʹ

and

reverse

190

5ʹ-GTGGTAACCGCTCAGGTGTTG-3ʹ)

191

5ʹ-GTGCTATGTTGCTCTAGACTTCG-3ʹ

192

5ʹ-ATGCCACAGGATTCCATACC-3ʹ). The relative fold of gene expression was

193

calculated by the 2-∆∆Ct method.

194

Statistics Analysis

195

All data was assessed using analysis of variance (ANOVA) with Dunnettʹs t-test for

196

multiple comparison of data.

197

RESULTS AND DISCUSSION

198

Structure Elucidation

and

β-actin and

10


(forward reverse

Page 11 of 37


199

The structures of compounds 1-5 (Figure 1) were determined according to 1H-NMR,

200

13

201

reported and compound 5 was isolated from P. oleracea for the first time.

C-NMR and extensive spectroscopic analyses; compounds 1-4 were not previously

202

Compound 1 was obtained as yellow amorphous powder, m.p. : 136.5-138 ºC,

203

which showed a yellow-brown fluorescence at 365 nm and turned red-brown when

204

exposed to Dragendorff reagent. the IR spectrum displayed the absorption rates

205

characteristic of an amino group (3354, 1554 cm-1) and a carbonyl group (1666 cm-1).

206

The molecular formula (C18H26N2O) with 7 degrees of unsaturation was deduced from

207

the

208

287.2118 [M + H]+. The 1H NMR,

209

resonances, including seven methyls, two methylenes, and nine quaternary carbons

210

including one carbonyl, five olefinic carbons and three aliphatic carbons. Additionally,

211

one active hydrogen (δH-18 4.07, 1H, bs) was observed. The 1H NMR (500 MHz,

212

CDCl3) and

213

(Figure 2) spectrum showed cross-peaks of H2-6/C-5, C-7, C-8, C-9, C-4a and C-14;

214

H2-9/C-6, C-7, C-8, C-10, C-16 and C-17; H3-14/C-6, C-7, C-8, C-9 and C-10;

215

H3-15/C-8 and C-8a; H3-16/C-9, C-10 and C-17; H3-17/C-9, C-10 and C-16,

216

indicating that there are two six-membered rings sharing three same carbon (C-7, C-8,

217

C-8a) atoms, among which N was connected with C-8a, C-10 and C-2 because of the

218

downfield

219

(C-16, C-17) were located at C-10, and methyl C-14 was located at C-7. The first ring

220

had two aliphatic carbons and four olefinic carbons; the second ring had three

13

C NMR and HR-ESI-TOF-MS with a protonated molecular ion peak at m/z

13

13

C NMR and DEPT spectra showed 18 carbon

13

C NMR (125 MHz, CDCl3) data are listed in Table 1. The HMBC

C NMR chemical shifts (δC-8a 141.0, δC-10 50.6, δC-2 65.7); two methyls

11



Page 12 of 37

221

aliphatic carbons and two olefinic carbons. The HMBC showed the cross-peaks of

222

H3-11/C-2, C-3 and C-12; H3-12/C-2, C-3 and C-11; and H3-13/C-3, C-4 and C-4a,

223

suggesting a third six-membered ring fragment, sharing three identical (C-4a, C-8a, N)

224

atoms with the above two six-membered rings; two methyls (C-11, C-12) were

225

located at C-2; methyl C-13 was located at C-4. To satisfy the molecular formula, the

226

presence of the imino group at C-5 was reasonable considering the downfield

227

chemical shift of C-5 (δC 169.4). The overall structure of compound 1 was further

228

confirmed by additional 2D NMR data (Figure 1). The relative configuration was

229

confirmed by the NOE spectrum, according to the correlations of H3-14/H3-16, Ha-6/

230

H3-14, Ha-9/ H3-13, Ha-9/ H3-14 and Ha-9/ H3-16, as illustrated in Figure 3. Thus, the

231

structure

232

5-imino-2,2,4,7,8,10,10-heptamethyl-6,7-dihydro- 2H-1,7-ethanoquinolin-3(5H)-one,

233

named oleracimine, 1.

of

compound

1

was

elucidated

as

234

Compound 2 was obtained as a yellow amorphous powder, m.p. : 148.0-149.5 ºC,

235

which showed a yellow fluorescence at 365 nm and turned pink when treated with

236

Dragendorff reagent.the IR spectrum suggested the existence of amino group (3470,

237

3290 and 1578 cm-1) and two carbonyl (1712 and 1668 cm-1) groups. The molecular

238

formula (C18H24N2O2) with 8 degrees of unsaturation was deduced from the

239

HR-ESI-TOF-MS with a protonated molecular ion peak at m/z 301.1912 [M + H]+.

240

The 1H NMR, 13C NMR and DEPT spectra showed 18 carbon resonances, including

241

six methyls, one methylene, one methine, and ten quaternary carbons including two

242

carbonyl groups, five olefinic carbons and three aliphatic carbons. Three active 12


Page 13 of 37


243

hydrogens were also observed. 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz,

244

CDCl3) data are provided in Table 1. The HMBC (Figure 2) spectrum of compound 2

245

showed cross-peaks of H2-10/C-1, C-2, C-7, C-8a, C-9 and C-16; H1-3a/C-8, C-8a;

246

H3-11/C-2, C-3 and C-12; H3-12/C-2, C-3 and C-11; H3-16/C-1, C-2, C-8a and C-10,

247

indicating that there were two carbon rings sharing the same two carbon (C-1 and

248

C-8a) atoms, two methyls (C-11 and C-12) located at C-3 which was connected to C-2,

249

and one methyl (C-16) located at C-1 which was connected to C-2, C-8a, and C-10.

250

The first ring had two aliphatic carbons and four olefinic carbons; the second ring had

251

three aliphatic carbons, one olefinic and one carbonyl carbon. The HMBC cross-peaks

252

of H1-3a/C-5; H3-13/C-4, C-5 and C-14; H3-14/C-4, C-5 and C-13; and H3-15/C-5,

253

C-6 and C-7 suggested that there was a seven-membered ring sharing at least three

254

carbons (C-3a, C-7 and C-8a) atoms with the above two rings, two methyls (C-13 and

255

C-14) linked to C-4 which was connected to C-5, and one methyl (C-15) located at

256

C-6 which was connected to C-7 and C-5. To satisfy the molecular formula, the

257

presence of the imino group at C-9 was reasonableconsidering the downfield chemical

258

shift of C-9 (δC 166.8). In addition, the presence of the amino group was located at

259

C-8 because of the downfield 1H and 13C NMR chemical shifts (δC-8 142.0), and C-8

260

was located between quaternary carbons C-7 and C-8a considering the only

261

cross-peak of H1-3a/C-8. The complete structure of compound 2 was further confirmed

262

by additional 2D NMR data (Figure 1). In addition, the ultraviolet absorptions,

263

infrared absorptions and NMR data of compound 2 were of high similarity to

264

compound 1, which also verified the above structure of compound 2. The relative 13



Page 14 of 37

265

configuration was confirmed by NOE spectrum, as indicated by correlations of

266

H1-3a/H3-11, H3-12/H3-16 and Hb-10/16, as illustrated in Figure 3. Therefore, the

267

structure

268

8-amino-9-imino-1,3,3,4,4,6-hexamethyl-3a,4-dihydro-1,7-ethanoazulene-2,5(1H,3H)

269

-dione, named oleracimine A, 2.

of

the

compound

2

was

elucidated

as

270

Compound 3 was obtained as a brown crystal, m.p. : 121.5-122.5 ºC, which showed

271

blue fluorescence at 365 nm and turned orange when exposed to Dragendorff reagent.

272

The IR spectrum displayed the absorption characteristic of the amino groups (3330

273

and 3278 cm-1) and two carbonyl groups (1650 and 1620 cm-1). The molecular

274

formula (C18H28N2O2) with 6 degrees of unsaturation was deduced from the

275

HR-ESI-TOF-MS with a protonated molecular ion peak at m/z 305.2359 [M + H]+.

276

The 1H NMR, 13C NMR and DEPT spectra showed 18 carbon resonances, including

277

seven methyls, one methylene, three methines, and seven quaternary carbons

278

including two carbonyl groups, four olefinic carbons and two aliphatic carbons. Two

279

active hydrogens were also observed. The 1H NMR (500 MHz, CDCl3) and 13C NMR

280

(125 MHz, CDCl3) data are listed in Table 1. The HMBC (Figure 2) spectrum showed

281

cross-peaks of H1-2'/C-3', C-4', C-9'a, C-10' and C-15'; H2-4'/C-3', C-5', C-6', C-10'

282

and C-11'; H-6'/C-4', C-6'a, C-9'a; H1-6'a/C-5' and C-6'; H3-15'/C-1' and C-9'a,

283

indicating that there is one eight-membered ring including a secondary amino group

284

located between C-1' and C-3' and that H1-2' (δH 4.61) belonged to the secondary

285

amino group. Two methyls (C-10' and C-11') were located at C-3', and one methyl

286

(C-15') was located at C-1'. The active hydrogen (δH 5.40) was part of the secondary 14


Page 15 of 37


287

amino group located at C-5' because of the downfield 13C NMR chemical shifts (δC-1'

288

152.7, δC-3' 60.0, and δC-5' 132.0). In addition, the HMBC spectrum showed

289

cross-peaks of H3-2/C-1, and the cross-peaks of active hydrogen (δH 5.40)/C-1 and

290

C-2 indicated that an acetamide existed. Correlations of active hydrogen (δH

291

5.40)/C-6'a and C-7' were also observed in the HMBC spectrum. The cross-peaks of

292

H1-6'a/C-7', C-8', C-9', C-9'a, C-12' and C-13'; H1-9'/C-6', C-6'a, C-7', C-8', C-9'a and

293

C-14'; H3-10'/C-3', C-4', and C-11'; H3-11'/C-3', C-4' and C-10'; H3-12'/C-6'a, C-7' and

294

C-13'; H3-13'/C-6'a, C-7' and C-12'; H3-14'/C-8' and C-9' indicate the existence of a

295

cyclopentane with one carbonyl (δC-8' 201.0) and three methyls including two methyls

296

(C-12', C-13') located at C-7' and one methyl (C-14') located at C-9', sharing two

297

carbon atoms (C-9'a, C-6'a) with the above ring. The overall structure was further

298

confirmed by additional 2D NMR data (Figure 1). The NOE spectrum suggested the

299

correlations of H3-14'/H3-15' and H1-9'; H3-15'/H3-10'; H3-12'/H1-6'a, 6', 9';

300

H1-9'/H1-6'a and H3-14'; and H1-6'a/H-6', as illustrated in Figure 3. Therefore, the

301

structure

302

N-((1E,5E)-1,3,3,7,7,9-hexamethyl-8-oxo-3,4,6a,7,8,9-hexahydro-2H-cyclopenta[c]az

303

ocin-5-yl)acetamide and was named oleracone A, 3.

of

compound

3

was

elucidated

as

304

Compound 4 was obtained as an colorless oil, which showed blue fluorescence at

305

365 nm, The IR spectrum suggesting that there is one hydroxyl group (3420 and 1310

306

cm-1) and one carbonyl group (1760 cm-1). The molecular formula (C13H18O2) with 5

307

degrees of unsaturation was deduced from the HR-ESI-TOF-MS with a protonated

308

molecular ion peak at m/z 207.1389 [M + H]+. The 1H NMR, 15


13

C NMR and DEPT


Page 16 of 37

309

spectra showed 18 carbon resonances, including three methyls, three methylenes and

310

two methines, and five quaternary carbons including one carbonyl, two olefinic

311

carbons and two aliphatic carbons. The 1H NMR (500 MHz, CDCl3) and

312

(125 MHz, CDCl3) data are listed in Table 1. The HMBC (Figure 2) spectrum showed

313

cross-peaks of H-1/C-3a and C-8; Ha-2/C-1, C-3a, C-8 and C-8a; H3-9/C-2, C-3, C-3a

314

and C-10; H3-10/C-3, C-3a and C-9; H2-4/C-3, C-3a and C-5; Hb-6/C-4, C-7, C-8 and

315

C-11; H-8/C-1, C-3a, C-6, C-8a and C-11; H3-11/ C-6, C-7 and C-8. The 1H−1H

316

COSY cross-peaks of H-1/Ha-2 and Hb-4; Ha-4/H3-9 and H3-10; Ha-6/H-8; H-8/H3-11;

317

H3-9/H3-10. The HMBC and 1H-1H COSY indicated the existence of a polysubstituted

318

azulene with one carbonyl carbon at C-5, two methyls (C-9, C-10) at C-3, one methyl

319

(C-11) at C-7, and one hydroxy at C-3a, considering the downfield chemical shift of

320

C-3a (δC 71.3). The overall structure of compound 4 was further confirmed by

321

additional 2D NMR data (Figure 1). The relative configuration was confirmed by

322

NOE spectrum, in which existed the correlation of H-8/H3-11 and H-1; Ha-4/H3-10;

323

Hb-4/H3-9; Ha-2/H3-10; and Hb-2/H3-9, as illustrated in Figure 3. Therefore, the

324

structure

325

3a-hydroxy-3,3,7-trimethyl-2,3a,4,6-tetrahydroazulen-5(3H)-one and was named

326

oleracone B, 4.

of

compound

4

was

elucidated

13

C NMR

as

327

Compound 5 was obtained as an colorless color, needle crystal, m.p. : 198-199 ºC,

328

which showed a blue-violet fluorescence at 365 and 254 nm and turned red-brown

329

when exposed to Dragendorff reagent. The molecular formula (C11H8N2) with 9

330

degrees of unsaturation was deduced from the HR-ESI-TOF-MS with a protonated 16


Page 17 of 37


331

molecular ion peak at m/z 169.0777. The structure was further confirmed by

332

additional NMR data. All the spectroscopic data revealed that compound 5

333

corresponded to reported values of β-carboline (Figure 1) in the literature. 22-24

334

Assay of 1 for Anti-inflammation

335

Cell Viability

336

The cytotoxic effect of 1 on RAW 264.7 cells was observed at a concentration of 50

337

µM by MTT assays (Figure 4). Therefore, concentrations between 1-20 µM were

338

considered non-cytotoxic and were used for subsequent experiments.

339

Effects of Oleracimine, 1, on Inflammatory Mediators

340

Figure 5 shows the results of oleracimine, 1, against the production of NO (Figure. 5A)

341

in LPS-induced RAW 264.7 cells. Oleracimine, 1, suppressed the secretion of

342

inflammatory mediators in a dose-dependent manner because 4 µM for NO and PGE2

343

(Figure 5D) and 10 µM for IL-6 (Figure 5B) and TNF-α (Figure 5C) began to reveal

344

significant differences in comparison with the LPS group. A similar effect with 20

345

µM oleracimine was also observed in Dex and NS-398 treated cells.

346

Effects of Oleracimine, 1, on Protein and mRNA Expression

347

The concentration-dependent anti-inflammatory function of oleracimine, 1, on the

348

protein and gene levels is shown in Figure 6. Immunoblots revealed that LPS induced

349

significant upregulation in the protein expression of iNOS (Figure 6A) and COX-2

350

(Figure 6B) compared with the control group; however, oleracimine, 1,

351

down-regulated functions (Figure 6C). Furthermore, the mRNA expression levels of

352

iNOS (Figure 6D) and COX-2 (Figure 6E) followed the same as the trends of the 17



353 354

Page 18 of 37

proteins. Four novel compounds were isolated from P. oleracea; the structures were 1

elucidated according to HR-ESI-TOF-MS,

356

spectroscopic analysis techniques. Considering the anti-inflammatory activities of the

357

alkaloids, oleracimine, 1, was found in relative high-abundance and was selected to

358

investigate anti-inflammatory properties.

359

H-NMR,

13

355

C-NMR and extensive

Inflammation, as a manifestation of a complex immune response initiated by 25, 26

360

foreign invasion or tissue injury,

361

atherosclerosis, Alzheimerʹs disease, Parkinsonʹs disease, rheumatoid arthritis and

362

diabetes mellitus.

363

mediators, including cytokines, nitric oxide and enzymes that are released when

364

macrophages undergo different phenotypes against stimuli.

365

tumor necrosis factor (TNF) and interleukins (IL), modulate the host defense

366

mechanism stimulated by unknown causes or inflammatory diseases and are

367

characteristic products during an inflammatory process.

368

catalyzed by the inducible nitric oxide synthase (iNOS), was regarded as a vital

369

mediator in the process and was reported to affect the products of the cyclooxygenase

370

metabolic pathway with two cyclooxygenase isoforms, cyclooxygenase-1 (COX-1)

371

and cyclooxygenase-2 (COX-2).

372

metabolites predominantly catalyzed by COX-2, takes part in the processes of classic

373

inflammatory symptoms, such as swelling and pain.

374

LPS-induced RAW 264.7 cells were selected as the experimental model because the

27, 28

is associated with various diseases, such as

The effect occurs in association with many inflammatory

32, 33

29, 30

31

Cytokines, such as

Nitric oxide (NO),

Prostaglandins E2 (PGE2), one of the

18


34

Thus, in this study,

Page 19 of 37


375

macrophages could sensitively respond to the bacterial endotoxin, lipopolysaccharide,

376

which is the common stimuli to establish the experimental model of inflammation. 35,

377

36

378

regulate the immune system against pathogens.

379

ensured that it was possible to determine the immunoregulatory extent of oleracimine,

380

1. Cytokines function in immune system regulation, cell proliferation and

381

inflammation and are related to the expression of iNOS, which remains stable at the

382

mRNA and protein levels. Additionally, iNOS plays a crucial role in producing NO

383

during the inflammatory process.

384

PGs, and COX-1 is predominantly involved in homeostatic functions, whereas COX-2

385

is activated by inflammatory stimuli in the pathophysiology of inflammatory diseases

386

to produce excessive PGs. 40 In this study, the LPS group exhibited overexpression of

387

the

388

dose-dependently decrease the secretions of IL-6, TNF-α, NO and PGE2 in culture

389

supernatants and could decrease the expression of protein and mRNA levels of iNOS

390

and COX-2; however, the inhibitory features of oleracimine, 1, had nothing to do with

391

to its cytotoxicity because the MTT assays aforementioned showed no effects on the

392

viability of LPS-induced cells at the corresponding concentrations. In Figure 6, the

393

protein expression and the mRNA levels of iNOS and COX-2 are decreased at 1 µM;

394

although, the variation does not reflect on the mediators in the identical

395

concentrations. Except for iNOS and COX-2, multi-enzyme participation in

396

inflammatory signaling pathways contributes to the secretion of mediators, which

Stimulated macrophages could secrete various proinflammatory mediators to

tested

mediators,

38, 39

whereas

37

The production of these mediators

COX-1 and COX-2 catalyze the generation of

pretreatment

with

19


oleracimine,

1,

could


Page 20 of 37

397

leads to an asynchronous tendency between the enzymes and the catalytic products.

398

However, the asynchronous tendency provides an orientation for future research.

399

Collectively, these results indicate that oleracimine, 1, is a promising active

400

anti-inflammatory compound.

401

It is all known that P. oleracea being as wild vegetable, is commonly used in daily

402

consumption. Furthermore, we found that micromole level of oleracimine, 1, can

403

present remarkable anti-inflammation activity. Therefore, sufficient P. oleracea

404

consumed as food can exert the anti-inflammatory action especially synergistic effect

405

with other anti-inflammatory compounds. In a word, P. oleracea, being an important

406

vegetable crop with a variety of components that have multiple health functions, will

407

draw increasingly more attention from food scientists because it is ubiquitous

408

worldwide and has abundant nutritional and pharmaceutical values.

409

ABBREVIATIONS

410

TNF-α, tumor necrosis factor alpha; IL-6, interleukins-6; NO, nitric oxide; iNOS,

411

inducible

412

cyclooxygenase-2; PGE2, prostaglandins E2; LPS,

413

dexamethasone; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide;

414

TBST, Tris-buffered saline with Tween; PBS, phosphate-buffered saline; PMSF,

415

phenylmethylsulfonyl fluoride; PVDF, polyvinylidene fluoride;

416

FUNDING SOURCES

417

This work was funded by a project of the National Natural Science Foundation of

418

China (Grant No. 81573546) and the Natural Science Foundation of Liaoning

nitric

oxide

synthase;

COX-1,

cyclooxygenase-1;

COX-2,

lipopolysaccharide; Dex,

20


Page 21 of 37


419

Province (Grant No. 2015020699), China.

420

SUPPORTING INFORMATION

421

Spectroscopic and spectrometric data for compounds 1-5. This material is available

422

free of charge via the Internet at http://pubs.acs.org.

423 424 425 426 427 428 429 430

21



431

Page 22 of 37

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544

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27



562

FIGURE CAPTIONS

563

Figure 1. Structures of compounds isolated from Portulaca oleracea. 1: oleracimine,

564

2: oleracimine A; 3: oleracone A; 4: oleracone B; 5: β-carboline; 6: oleracone

565

(previously isolated alkaloid in our laboratory).

566

Figure 2. Selected 1H-1H COSY and HMBC correlations of compounds 1-4.

567

Figure 3. Selected 1H-1H NOESY correlations of compounds 1-4.

568

Figure 4. Cell viability of the LPS-induced macrophage RAW 264.7 cells pretreated

569

with oleracimine, 1. The data represent the mean ± SD of three independent

570

experiments. *p ＜ 0.05 vs. con group; #p ＜ 0.05 vs. LPS group.

571

Figure 5. Inhibitory effect on (A) NO, (B) IL-6, (C) TNF-α and (D) PGE2 production

572

in the culture media of LPS-induced RAW 264.7 cells pre-incubated with oleracimine.

573

Dexamethasone and NS-395 were used as positive control. The data represent the

574

mean ± SD of three independent experiments. *p ＜ 0.05 vs. con group; #p ＜

575

0.05 vs. LPS group.

576

Figure 6. Inhibitory effect on the proteins and genes of COX-2 and iNOS production

577

in the LPS-induced RAW 264.7 cells. Proteins: (A) iNOS, (B) COX-2, (C) western

578

blot; genes: (D) iNOS and (E) COX-2. The proteins were detected by band densities.

579

The mRNA was calculated using the 2-∆∆Ct method. The data represent the mean ±

580

SD of three independent experiments. *p ＜ 0.05 vs. con group; #p ＜ 0.05 vs. LPS

581

group.

582

28


Page 28 of 37

Page 29 of 37


Table 1.

position

δC, type

1

H-NMR (500 MHz) and 13C-NMR (125 MHz) Data of Compounds 1-4 in CDCl3

1 δH, mult (J δC, type in Hz) 49.3, C 212.1, C

2 δH, mult (J δC, type in Hz) 122.1, CH 27.5, CH2

1 2

N 65.7, C

3 3a 4

206.2, C 121.3, C

60.2, C 22.1, CH 65.9, C

4a 5 6

143.5, C 169.4, C 52.3, CH2

206.7, C 122.2, C

199.7, C 27.6, CH2

7 8 8a 9

39.0, C 110.7, C 141.0, C 46.8, CH2

140.3, C 142.0, C 110.6, C 166.8, C

149.9, C 123.5, CH 154.1, C 24.4, CH3

10

50.6, C

11 12 13

27.4, CH3 29.1, CH3 14.4, CH3

2.56,d,(15.3) 2.28,d,(15.3)

2.02,d,(13.5) 1.47,d,(13.5)

43.3, CH2 1.45,s 1.31,s 1.88,s

28.4, CH3 27.6, CH3 27.8, CH3

2.02,s

2.85,d,(17.1) 2.60,d,(17.1) 1.37,s 1.49,s 1.44,s

39.8, C 71.3, C 49.1, CH2

4 δH, mult (J in Hz) 5.67,s 1.97,dd,(5.0,1.7) 1.75,dd,(5.0,1.7)

2.88,d,(15.0) 2.07,d,(15.0)

2.47,d,(5.0) 2.17,d,(5.0) 6.00,s 1.00,s

23.0, CH3

1.13,s

24.3, CH3

1.93,s

29


position

δC, type

1 2 1ʹ 2ʹ 3ʹ 4ʹ

169.5, C 24.6, CH3 152.7, C NH 51.0, C 42.7, CH2

5ʹ 6ʹ 6ʹa 7ʹ 8ʹ 9ʹ 9ʹa 10ʹ 11ʹ 12ʹ 13ʹ 14ʹ 15ʹ

132.0, C 112.0, CH 48.3, CH 58.1, C 200.6, C 44.0, CH 101.0, C 29.0, CH3 27.3, CH3 25.2, CH3 24.4, CH3 20.3, CH3 22.0, CH3 NH

3 δH, mult (J in Hz) 1.90,s 4.61,bs 2.33,d,(13.9) 2.19,d,(13.9) 5.05,d,(6.2) 2.92,d,(5.2)

2.44,q,(7.3) 1.29,s 1.08,s 1.39,s 1.09,s 1.08,d,(7.3) 2.35,s 5.40,bs


14 15 16 17 18

28.8, CH3 21.2, CH3 28.6, CH3 32.5, CH3 NH

1.17,s 1.82,s 1.34,s 1.30,s 4.07,bs

28.6, CH3 14.7, CH3 25.5, CH3 NH2 NH

1.34,s 1.95,s 1.31,s 1.62,bs 4.01,bs

30


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Figure 1.

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Figure 2.

32


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Figure 3.

33



Figure 4.

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Figure 5.

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Figure 6

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Table of Contents Graphic

37


Three Novel Alkaloids from Portulaca oleracea L ... - ACS Publications

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