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ANTI-INFALMMATORY PRINCIPLES FROM SARCANDRA GLABRA Yun-Chen Tsai, Shih-Han Chen, Lie-Chwen Lin, and Shu-Ling Fu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05125 • Publication Date (Web): 22 Jan 2017 Downloaded from http://pubs.acs.org on January 25, 2017

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Journal of Agricultural and Food Chemistry

Anti-inflammatory principles from Sarcandra glabra

Yun-Chen Tsai,† Shih-Han Chen,∥ Lie-Chwen Lin,*, †

∥

and Shu-Ling Fu*,†

Institute of Traditional Medicine, National Yang-Ming University, Taipei 11221 Taiwan

∥

National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 11221, Taiwan

Corresponding Author *(L. C. Lin) Phone: +886-2-2820-1999 (ext. 7101). Fax: +886-2-28204276. E-mail: [email protected]; *(S. L. Fu) Phone: +886-2-28267177. Fax: +886-2-28225044. E-mail: [email protected] Conflict of Interest The authors declare no competing financial interest.

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1

ABSTRACT

2

Sarcandra glabra (Thunb.) Nakai (Chloranthaceae) is a medicinal plant used

3

as herbal tea or food supplement to promote human health. We isolated 14 phenolic

4

compounds from the n-butanol fraction of S. glabra and investigated their

5

anti-inflammatory potential using lipopolysaccharide (LPS)-activated RAW264.7

6

macrophages. We demonstrated that methyl isorinate, a previously uncharacterized

7

compound in S. glabra, is able to suppress NF-κB activation and reduce the

8

expression of iNOS and COX-2 as well as the phosphorylation of IκB in

9

LPS-treated RAW264.7 cells. In addition, the production of two inflammatory

10

cytokines (IL-6 and TNF-α), as well as release of reactive oxygen species, in the

11

LPS-stimulated macrophages, was also inhibited by this compound. Furthermore,

12

the structure-and-activity relationship of all of the isolated phenolic compounds

13

present were analyzed. Overall, this study revealed several anti-inflammatory

14

compounds that were present in S. glabra and our results suggest that these diverse

15

phenolic compounds are associated with the anti-inflammatory effects of S. glabra.

16 17

Key words: anti-inflammation, Sarcandra glabra, methyl isorinate, NF-κB

18


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INTRODUCTION

20

Sarcandra glabra (Thunb.) Nakai (Chloranthaceae) is distributed in the

21

southern Asia and has been used as folk medicine for treating diseases such as

22

cancer, inflammation, diarrhea, and rheumatism.1 It is also used as herbal tea and

23

food supplement in China to treat various ailments, to enhance mental efficiency and

24

to lessen stress/weakness.2 Previous publications have shown that S. glabra displays

25

anti-inflammatory activity, anti-viral activity, cytoprotective activity and anticancer

26

properties.1 The chemical constituents identified from this plant include

27

sesquiterpenes, phenolic acid, flavonoids, chalcones, polysaccharides, coumarins

28

and triterpenoids.3-8 The bioactivities of some of these compounds have been

29

reported

30

immune-regulation and anticancer properties.3, 9-11

previously

and

include

anti-oxidation,

anti-inflammation,

31

Aberrant inflammation promotes the progression of a variety of diseases

32

including Alzheimer’s disease, inflammatory bowel disease and arthritis.12

33

Macrophages play a central role in inflammatory diseases because they are able to

34

produce multiple pro-inflammatory molecules in response to a range of stimuli

35

including lipopolysaccharides (LPS).13 Nuclear factor κB (NF-κB) is a crucial

36

transcription factor when triggering an inflammatory reaction and is also known to

37

be a molecular target of anti-inflammatory drugs. Upon LPS stimulation, the 3



38

inhibitor of NF-κB, the IκB protein, is phosphorylated by I kappa B kinase;

39

following this it is degraded by proteasomes. In the absence of IκB, the p50/p65

40

heterodimer of NF-κB is able to enter the nucleus, and this lead to the transcriptional

41

activation of various downstream genes. NF-κB activation in turn results in the

42

upregulation of pro-inflammatory mediators, which include a number of cytokines

43

(e.g. TNF-α and IL-6), various enzymes (e.g. iNOS and COX-2), different adhesion

44

molecules, chemokines and reactive oxygen species (ROS).12

45

Although the anti-inflammatory potential of S. glabra has been previously

46

reported, the anti-inflammatory ingredients in S. glabra have not been fully explored.

47

In this study, we identified fourteen phenolic compounds in the n-butanol fraction of

48

S. glabra and further characterized their anti-inflammatory effect using

49

lipopolysaccharide (LPS)-induced RAW264.7 macrophages as the model system.

50

We found an anti-inflammatory compound, methyl isorinate. This compound is able

51

to significantly inhibit the activation of NF-κB, the expression of iNOS and COX-2

52

and the production of IL-6 and TNF-α, as well as reducing ROS production in

53

LPS-stimulated macrophages. We also explored the structure/activity relationships

54

among all of the isolated phenolic compounds obtained from S. glabra.

4


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MATERIALS AND METHODS

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Experimental Instruments. 1H-, 13C- and 2D NMR spectra were measured

57

on a Varian VNMRS600 spectrometer using deuterated solvents as the internal

58

standards. ESIMS data were recorded on a Finnigan LCQ spectrometer. Column

59

chromatography was carried out using Sephadex LH-20 (GE Healthcare Biosciences

60

AB, Sweden) and Silica gel 60 (70-230 or 230-400 mesh, Merck; or 12-26 µm,

61

Eurochrom, Knauer). TLC was conducted on precoated Kieselgel 60 F254 plates (0.2

62

mm, Merck). Silica gel 60 F254 (1 mm, Merck) was used for the preparative TLC.

63

Semipreparative HPLC analysis was performed using a Shimadzu LC-8A pump and

64

a SPD-10A vp UV-Vis detector. A Cosmosil 5C18-AR-II column (20 × 250 mm;

65

particle size 5 µm; Nacalai tesque, Kyoto, Japan) or a Beta Basic Cyano column (10

66

× 250 mm; particle size 5 µm, Thermo scientific, USA) were used for various

67

separations.

68

Plant Material. The aerial parts of Sarcandra glabra were collected from

69

Hsinchu County, Taiwan, in September 2015, and verified by comparisons with a

70

voucher specimen (NHP-00472) of S. glabra deposited at the Herbarium of the

71

National Research Institute of Chinese Medicine, Taipei, Taiwan.

72

Extraction and Isolation. Fresh aerial parts of S. glabra (10 kg) were cut

73

into pieces and extracted three times with EtOH (80 L) under reflux. After removal 5



74

of the solvent, the crude extract was separated into n-hexane, EtOAc, n-BuOH and

75

H2O layers using liquid-liquid partitioning. The n-BuOH fraction (42 g) was

76

subjected to column chromatography on Sephadex LH-20 (10 × 56 cm) and was

77

eluted with MeOH to give seven fractions (Frs. 1-7). Fraction 6 was crystallized

78

from EtOH to give 1 (1.01g). Fraction 5 (24 g) was rechromatographyed on

79

Sephadex-LH-20 (5 × 90 cm) and when eluted with EtOH; this gave eight

80

subfractions (Fractions 5-1 ~ 5-8). Fraction. 5-4 (3 g) was further separated on a

81

silica gel column (2.8 × 48 cm) followed by elution with a solvent mixture of

82

CHCl3/EtOAc/MeOH/H2O in the ratio of 4:4:2:1 (550 mL), and then with the same

83

solvent, mixture in the ratio 2:4:2:1 (900 mL); this gave ten subfractions (Fractions

84

5-4-1 ~ Fr. 5-4-10). Fractions 5-4-3 (266 mg), 5-4-4 (234 mg), and 5-4-5 (255 mg)

85

were individually chromatographed by semipreparative HPLC (Beta Basic Cyano

86

column using an eluting solvent system of MeOH/H2O; flow rate: 3.7 mL/min) to

87

give 2 (18 mg), 3 (10 mg), 5 (2 mg), 6 (6 mg), 8 (26 mg), 9 (5 mg), 10 (8 mg), 12 (2

88

mg), 13 (13 mg), and 14 (5 mg) from Fraction 5-4-3; 5 (26 mg), and 11 (8 mg) from

89

Fraction 5-4-4; and 7 (13 mg) from Fraction 5-4-5. Fraction 5-5 (1.51g) was

90

repeatedly chromatographed using a semipreparative HPLC (Cosmosil 5C18-AR-II

91

column using an eluting solvent system of MeOH/H2O; flow rate: 3.7 mL/min); this

92

was followed by preparative TLC (solvent system: CHCl3: EtOAc: MeOH: H2O = 6


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15: 40: 22: 10) and the products obtained were 1 (593 mg), 2 (36 mg), 3 (220 mg), 4

94

(14 mg), and 5 (14 mg).

95

Chemicals and Antibodies. Andrographolide, fisetin and albumin bovine

96

serum (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

97

Lipopolysaccharide (LPS) was purchased from InvivoGen (California, USA). The

98

antibodies against phospho-IκB-α (Ser 32), IκB-α and COX-2 were purchased from

99

Cell Signaling Technology (Danvers, MA), while anti-iNOS antibody was obtained

100

from Abcam (Cambridge, United Kingdom). Anti-β-actin antibody was obtained

101

from Sigma-Aldrich.

102

Cell Culture. Murine RAW 264.7 macrophages were purchased from the

103

Food Industry Research and Development Institute (Hsinchu, Taiwan) and they were

104

regularly maintained in DMEM (Gibco, Grand Island, NY) containing 10% bovine

105

calf serum (Sigma-Aldrich), 100 units/mL penicillin, 100 µg/mL streptomycin, 2

106

mM glutamine, 1 mM sodium pyruvate (Gibco) in a 5% CO2 humidified incubator

107

at 37°C. RAW 264.7/Luc-P1 cells, a LPS responsive cell clone that stably expresses

108

a reporter gene (pELAM1-Luc), were generated and cultured as described

109

previously.14 The vector pELAM1-Luc contains a NF-κB-responsive promoter

110

region of the endothelial leukocyte adhesion molecule I (ELAM1) controlling the

111

firefly luciferase gene. 7



112

Luciferase Reporter Assay. RAW 264.7/Luc-P1 cells (1.5 x 105 cells in

113

24-well plates) were treated with S. glabra extracts, pure compounds, a positive

114

control (andrographolide) or vehicle (0.1% DMSO) for 1 h, which was followed by

115

LPS (10 ng/mL) treatment for 24 h. The treated cells were collected, lysed and

116

analyzed by luciferase assay as described previously.14 The luminescence was

117

measured using an Infinite® 200 PRO (Tecan Group Ltd, Männedorf, Switzerland).

118

Enzyme-linked Immunosorbent Assay (ELISA). RAW 264.7 cells

119

(1.5x105 cells in 24-well plates) were treated with the various compounds, a positive

120

control (andrographolide) or vehicle (0.1% DMSO) for 1 h and then incubated with

121

LPS (10 ng/mL) for 24 h. The TNF-α and IL-6 production in the medium was

122

measured using appropriate ELISA kits (eBioscience, San Diego, CA).

123

MTT Assay. RAW 264.7 cells (104 cells/well in 96-well plates) were treated

124

with various compounds or vehicle (0.1%DMSO) for 24 h. MTT assays were carried

125

out as described previously.14

126

Western Blotting. RAW 264.7 cells were treated with methyl isorinate, a

127

positive control (andrographolide) or vehicle (0.1% DMSO) and then incubated with

128

LPS for 24 h. Protein extract (50 µg) from each sample was analyzed using

129

SDS-PAGE, followed by transfer and Western blotting using a selected range of

130

antibodies. The Western blot protocol has been described formerly.14 The images of 8


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the Western blots were quantified using the Image J program version 1.48 (NIH,

132

Bethesda, MD).

133

ROS Production. RAW 264.7 cells (5x105 cells/well in 6-well plates) were

134

treated with methyl isorinate, a positive control (fisetin) or vehicle (0.1%DMSO) for

135

1 h and then incubated with LPS (50 ng/mL) for 24 h. ROS production was

136

measured using flow cytometry as described previously.15

137

Statistical Analysis. The results are presented as means ± standard deviation

138

(SD) from at least three independent experiments. The data was analyzed using the

139

Student’s t test and a p value of < 0.05 was considered statistically significant.

140 141

RESULTS

142

Isolation and identification of anti-inflammatory compounds from

143

Sarcandra glabra. The fresh aerial parts of S. glabra were extracted with EtOH,

144

then the EtOH extract was partitioned successively between H2O and n-hexane; this

145

was followed by partitioning with EtOAc and n-BuOH. We found that the n-hexane,

146

EtOAc, and n-BuOH fractions of the ethanol extract were able to suppress

147

LPS-induced NF-κB activation in RAW264.7 macrophages without causing

148

cytotoxicity (Supporting Information Figure 1). In folk medicine, S. glabra is

149

prepared for use as a water decoction. The polarity of n-BuOH fraction is closest to 9



150

that of a water decoction compared to either the n-hexane or EtOAc fractions. Based

151

on the above reason we carried out our isolation of active compounds from the

152

n-BuOH fraction first. The n-BuOH extract was subjected to a combination of

153

Sephadex LH-20, silica gel, reverse phase C18, and Beta Basic Cyano

154

chromatography using various solvent systems (as described in the experimental

155

section) and the result was a series of phenolic compounds 1-14. Compounds 1-14

156

were then identified to be rosmarinic acid (1)16, methyl isorinate (2),17, 18, 19 methyl

157

rosmarinate (3),16 (2R)-methyl-3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate (4),20

158

isorinic acid (5),19 butyl rosmarinate (6),20 5-O-caffeoylshikimic acid (7),21

159

5-O-caffeoylshikimic acid methyl ester (8),22 caffeic acid (9),16 methyl caffeate

160

(10),23 (2R)-3-(4-hydroxyphenyl)-2-hydroxypropionic acid (11),24 (2R)-methyl-3-

161

(4-hydroxyphenyl)- 2-hydroxypropanoate (12),25 3,4-dihydroxybenzoic acid (13),26

162

and methyl 3,4-dihydroxybenzoate (14),27 by various spectrometric methods (1D,

163

2D NMR, MS, and optical rotation) and by comparing their spectral data with the

164

values available in the literature. The results for some of these compounds are

165

presented below.

166

Rosmarinic acid (1): [α]25D 102 (c 1.0, MeOH); 1H NMR (600 MHz,

167

methanol-d4) 2.93 (1H, dd, J = 14.4, 9.6 Hz, Ha-7), 3.08 (1H, dd, J = 14.4, 3.6 Hz,

168

Hb-7), 5.08 (1H, dd, J = 9.6, 3.6 Hz, H-8), 6.26 (1H, d, J = 16.2 Hz, H-8′), 6.62 (1H, 10


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dd, J = 8.4, 2.4 Hz, H-6), 6.66 (1H, d, J = 8.4 Hz, H-5), 6.75 (2H, m, H-6, -5′), 6.91

170

(1H, dd, J = 7.8, 2.4 Hz, H-6′), 7.02 (1H, d, J = 2.4 Hz, H-2′), 7.50 (1H, d, J = 16.2

171

Hz, H-7′);

172

115.5 (C-8′), 116.2 (C-5), 116.4 (C-5′), 117.5 (C-2), 121.7 (C-6), 122.9 (C-6′), 127.9

173

(C-1′), 130.9 (C-1), 144.9 (C-4), 146.0 (C-3), 146.7 (C-3′), 146.8 (C-7′), 149.4

174

(C-4′), 169.0 (C-9′), 176.9 (C-9); ESIMS m/z 359 [M − H] .

13

C NMR (150 MHz, methanol-d4) 38.7 (C-7), 77.3 (C-8), 115.1 (C-2′),

−

175

Methyl isorinate (2): [α]25D 39 (c 1.0, MeOH); 1H NMR (600 MHz,

176

methanol-d4) 3.08 (2H, m, H-7), 3.68 (3H, s, -OCH3), 5.20 (1H, dd, J = 7.6, 5.6 Hz,

177

H-8), 6.25 (1H, d, J = 15.6 Hz, H-8′), 6.72 (2H, d, J = 8.4 Hz, H-3, -5), 6.77 (1H, d,

178

J = 8.0 Hz, H-5′), 6.94 (1H, dd, J = 8.0, 2.0 Hz, H-6′), 7.03 (1H, d, J = 2.0 Hz, H-2′),

179

7.07 (2H, d, J = 8.4 Hz, H-2, -6), 7.54 (1H, d, J = 15.6 Hz, H-7′);

180

MHz, methanol-d4) 37.6 (C-7), 52.7 (-C=OOCH3), 74.6 (C-8), 114.1 (C-8′), 115.2

181

(C-2′), 116.2 (C-3, -5), 116.5 (C-5′), 123.2 (C-6′), 127.5 (C-1′), 128.0 (C-1), 131.5

182

(C-2, -6), 146.8 (C-3′), 147.9 (C-7′), 149.8 (C-4′), 157.5 (C-4), 168.3 (C-9′), 172.1

183

(C-9); ESIMS m/z 357 [M−H] ; HRESIMS m/z 357.0972 [M − H] .

−

13

C NMR (150

−

184

Isorinic acid (5): [α]25D 114 (c 0.84, MeOH); 1H NMR (600 MHz, methanol-

185

d4) 2.98 (1H, dd, J = 14.4, 9.0 Hz, Ha-7), 3.13 (1H, dd, J = 14.4, 2.4 Hz, Hb-7), 5.09

186

(1H, dd, J = 9.0, 2.4 Hz, H-8), 6.25 (1H, d, J = 15.6 Hz, H-8′), 6.68 (2H, d, J = 8.4

187

Hz, H-3, -5), 6.76 (1H, d, J = 7.8 Hz, H-5′), 6.90 (1H, dd, J = 7.8, 1.8 Hz, H-6′), 11



188

7.02 (1H, s, H-2′), 7.12 (2H, d, J = 8.4 Hz, H-2, -6), 7.50 (1H, d, J = 15.6 Hz, H-7′);

189

13

190

114.6 (C-3, -5), 115.0 (C-5′), 121.4 (C-6′), 126.5 (C-1′), 129.0 (C-1), 129.9 (C-2, -6),

191

145.2 (C-7′), 145.3 (C-3′), 147.9 (C-4′), 155.5 (C-4), 167.6 (C-9′), 177.5 (C-9);

192

ESIMS m/z 343 [M − H] ; HRESIMS m/z 343.0816 [M − H] (calcd for C18H15O7,

193

343.0812).

C NMR (150 MHz, methanol-d4) 37.2 (C-7), 77.7 (C-8), 113.6 (C-2′), 114.2 (C-8′),

−

−

194

5-O-caffeoylshikimic acid (7): [α]25D −120 (c 1.0, MeOH); 1H NMR (600

195

MHz, methanol-d4) 2.31 (1H, dd, J = 18.0, 6.0 Hz, Ha-6), 2.88 (1H, dd, J = 18.0, 4.8

196

Hz, Hb-6), 3.87 (1H, dd, J = 7.8, 4.8 Hz, H-4), 4.37 (1H, br. s, H-3), 5.24 (1H, td, J =

197

6.0, 4.8 Hz, H-5), 6.27 (1H, d, J = 16.2 Hz, H-8′), 6.74 (1H, br. s, H-2), 6.77 (1H, d,

198

J = 7.8 Hz, H-5′), 6.94 (1H, dd, J = 7.8, 1.8 Hz, H-6′), 7.03 (1H, d, J = 1.8 Hz, H-2′),

199

7.55 (1H, d, J = 15.6 Hz, H-7′); 13C NMR (150 MHz, methanol-d4) 30.1 (C-6), 67.5

200

(C-3), 70.5 (C-4), 71.6 (C-5), 115.1 (C-8′), 115.2 (C-2′), 116.5 (C-5′), 123.0 (C-6′),

201

127.7 (C-1′), 132.8 (C-1), 136.3 (C-2), 146.8 (C-3′), 147.1 (C-7′), 149.6 (C-4′),

202

168.8 (C-7), 171.7 (C-9′); ESIMS m/z 335 [M − H] .

−

203

5-O-caffeoylshikimic acid methyl ester (8): [α]25D −80 (c 1.0, MeOH); 1H

204

NMR (600 MHz, methanol-d4) 2.32 (1H, dd, J = 18.0, 5.4 Hz, Ha-6), 2.84 (1H, dd, J

205

= 18.0, 3.0 Hz, Hb-6), 3.74 (3H, s, -OCH3), 3.91 (1H, dd, dd, J = 0.8, 4.2 Hz, H-4),

206

4.40 (1H, br. s, H-3), 5.23 (1H, dd, J = 5.4, 2.4 Hz, H-5), 6.25 (1H, d, J = 15.6 Hz, 12


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H-8′), 6.76 (1H, d, J = 7.8 Hz, H-5′), 6.84 (1H, br. s, H-2), 6.93 (1H, dd, J = 7.8, 1.8

208

Hz, H-6′), 7.03 (1H, d, J = 1.8 Hz, H-2′), 7.54 (1H, d, J = 15.6 Hz, H-7′); 13C NMR

209

(150 MHz, methanol-d4) 29.0 (C-6), 52.5 (-C=OOCH3), 67.2 (C-3), 69.8 (C-4), 71.2

210

(C-5), 115.0 (C-8′), 115.2 (C-2′), 116.5 (C-5′), 123.1 (C-6′), 127.7 (C-1′), 129.7

211

(C-1), 139.2 (C-2), 146.8 (C-3′), 147.3 (C-7′), 149.6 (C-4′), 168.3 (C-7), 168.6

212

(C-9′); ESIMS m/z 349 [M − H] .

−

213

Among the identified compounds, methyl isorinate (Figure 1A; compound 2),

214

was found to significantly inhibit LPS-induced NF-κB activation (Supporting

215

Information Table 1). The anti-inflammatory activity of methyl isorinate (2) has

216

never been reported previously and therefore the anti-inflammatory activity of this

217

compound was further characterized.

218

Methyl isorinate inhibits NF-κB activity in LPS-induced macrophages.

219

The inhibition of NF-κB activation by methyl isorinate in LPS-stimulated

220

RAW264.7/Luc-P1 macrophages was found to be concentration-dependent (Figure

221

1B). Consistent with this observation, methyl isorinate also decreased the amount of

222

phosphorylated IκB (the negative regulator of NF-κB) in a concentration-dependent

223

manner (Figure 1C). Notably, methyl isorinate did not show significant cytotoxicity

224

against RAW264.7 macrophages after 24-h treatment (Figure 1D).

225

Methyl isorinate suppresses the expression of pro-inflammatory 13



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molecules in LPS-stimulated RAW264.7 cells. TNF-α, IL-6, iNOS and COX-2 are

227

pivotal pro-inflammatory mediators during the inflammatory process.28 Next we

228

investigated whether methyl isorinate is able to reduce these pro-inflammatory

229

molecules induced by LPS. The results showed that methyl isorinate is able to

230

significantly suppress the LPS-induced production of TNF-α and IL-6 (Figure 2A).

231

Furthermore, the expression levels of iNOS and COX-2 were also reduced by

232

treatment with methyl isorinate (Figure 2B).

233

Methyl isorinate reduces LPS-triggered reactive oxygen species (ROS)

234

production in RAW264.7 macrophages. LPS is also an inducer of ROS production

235

in macrophages.29, 30 Therefore, we measured the effect of methyl isorinate on ROS

236

production in LPS-induced RAW264.7 macrophages using the fluorescent dye

237

H2DCFDA. As shown in Figure 3, methyl isorinate is able to suppress ROS

238

production in a concentration-dependent manner.

239

The structure-and-activity relationship analysis of the phenolic

240

compounds from S. glabra. In addition to methyl isorinate (2), which were

241

demonstrated to exhibit anti-inflammatory effects, we also investigated the

242

anti-inflammatory potential of other phenolic compounds isolated from S. glabra.

243

The inhibitory effects of these compounds on LPS-triggered NF-κB activation and

244

pro-inflammatory

cytokine

production

(IL-6

and

TNF-α)

14


in

RAW264.7

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245

macrophages were measured. As shown in Figure 4, rosmarinic acid (1) and its

246

structural analogs (2, 3, 5 and 6) were also able to suppress NF-κB activation and

247

IL-6 production, as well as exhibiting a trend suggesting inhibition of TNF-α

248

production. Furthermore, caffeic acid (9) and its structurally similar compounds (4,

249

10) also inhibited NF-κB activation, while compounds 9 and 10 were able to

250

reduced IL-6 expression; moreover, compound 4 could also reduce TNF-α

251

expression (Figure 5). On the other hand, compounds 11 and 12 had no effects on

252

NF-κB activity, on IL-6 production or on TNF-α expression. As for the shikimic

253

acid analog compounds 7 and 8, they significantly suppressed both NF-κB

254

activation and expression of the cytokines IL-6 and TNF-α (Figure 6). Finally, as

255

shown in Figure 7, the benzoic acid compounds 13 and 14 both impaired NF-κB

256

activation and IL-6 production, while at the same time showing an inhibitory trend

257

in terms of TNF-α production.

258 259

DISCUSSION

260

In this study, the anti-inflammatory activity of methyl isorinate, isolated from

261

S. glabra, was demonstrated; this compound in LPS-treated macrophages is able to

262

inhibit LPS-induced NF-κB activity and reduce IκB-α phosphorylation at non-toxic

263

concentrations (Figure 1). The compound is also able to suppress in the same system 15



264

the production of pro-inflammatory cytokines (Figure 2A), the expression of the

265

inflammatory enzymes (iNOS and COX-2; Figure 2B) and the production of ROS

266

(Figure 3). The anti-inflammatory potential of other structurally related phenolic

267

compounds isolated from S. glabra was also examined. Except for compounds 11

268

and 12, all of the other compounds were able to significantly inhibited NF-κB

269

activation and most of them showed a notable inhibitory trends regarding the

270

inhibition of IL-6/TNF-α production (Figures 4-7). In general, their inhibitory

271

effects on NF-κB activation is correlated with their capacities to reduce cytokine

272

production (IL-6 and TNF-α). Notably, our study, for the first time, demonstrates

273

that methyl isorinate (2) and three other phenolic compounds (5, 6 and 8) are

274

valuable anti-inflammatory compounds present in S. glabra. We have also measured

275

the bioactivities of a mixture of 1 and 3 in a ratio of 22:1 (Supporting Information

276

Fig 2 and Table 2), the ratio being based on their amount in the n-butanol fraction.

277

As shown in Supporting Information Fig 3 and 4, the activity of the major

278

compounds (1+3) present as a mixture is unable to account for the full activity of the

279

n-butanol fraction of S. glabra; thus it seems likely the other components present in

280

the S. glabra, such as compound 2, contribute to the inflammatory activity of this

281

plant. In addition, compounds 7, 9, 10, 13, at high concentrations, have been

282

demonstrated to exhibit anti-inflammatory activity,31-37 although the amounts of 16


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these compounds present in S. glabra are relatively low as compared with

284

compounds 1 and 3. Nevertheless, it is possible that these compounds may also be

285

involved in the anti-inflammatory activity of S. glabra. Taken together, our results

286

suggest that phenolic compounds play a major role in the ant-inflammatory effect of

287

S. glabra. The bioavailability and anti-inflammatory efficacy of these newly

288

identified phenolic compounds certainly merit further investigation using an animal

289

model.

290

In this study, we have demonstrated that methyl isorinate is able to suppress

291

the NF-κB pathway (Figure 1). A recent study has shown that methyl rosmarinate (3)

292

inhibits NF-κB activity in LPS-induced RAW264.7 cells via a stimulation of the

293

HO-1 signaling pathway and an inhibition of the MyD88 signaling pathway.31

294

Considering the structural similarity between methyl rosmarinate and methyl

295

isorinate, we speculated that methyl isorinate may inhibit NF-κB activity via the

296

HO-1 and MyD88-dependent pathways. However, this molecular event is needed to

297

be further studied.

298

Phenolics are a heterogenic group of compounds and include cinnamic acids,

299

flavonoids, and benzoic acids; they are produced as secondary metabolites by plants.

300

Recent reports have indicated that phenolic compounds are able to ameliorate

301

chronic inflammatory diseases such as diabetes, cardiovascular diseases, and 17



302

Alzheimer’s disease.38 In Figure 4, the findings related to the rosmarinic acid-related

303

compounds 1, 3, and 6 suggest that the carbon length of the alkyl ester seems to be

304

associated with their activity regarding cytokine inhibition. Moreover, rosmarinic

305

acid (1) has a caffeoyl group coupled with a phenyl propanoic acid and this

306

combination displays a stronger inhibition of NF-κB than the monomer compounds

307

(Figures 4 and 5). Therefore, both the carbon length of alkyl ester and the

308

dimerization of caffeic acid with phenyl propanoic acid appear to be important to the

309

anti-inflammation activity of the phenolic compounds described in this study. Based

310

on our findings presented in Figure 5, the presence of the methyl ester and the

311

double bond (C-7 and C-8) in caffeic acid analog (compound 10) would seem to

312

significantly increase its ability to suppress both NF-κB and IL-6 activity. Notably,

313

the replacement of an acid group with a methyl ester in the shikimic acid analogs did

314

not significantly affect their inhibitory activity (Figure 6).

315

Many plant-derived compounds exhibit immune-modulating activities and

316

also show satisfactory safety.39,40 Previously, several constituents of S. glabra,

317

namely caffeic acid, isofraxidin and rosmarinic acid, have been suggested as

318

antioxidants and anti-inflammatory agents.2 Moreover, S. glabra has been

319

previously shown to be non-toxic in vivo.41 Our study has further demonstrated that

320

the diverse phenolic compounds present in S. glabra show significant 18


Page 18 of 40

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321

anti-inflammatory potential. Thus, S. glabra is a valuable natural resource rich in

322

beneficial bioactive compounds and it can be further developed as a functional food.

323 324

Supporting Information

325

Supplemental tables: Compounds isolated from S. glabra and their effects on

326

NF-κB activity in LPS-stimulated RAW 264.7/Luc-P1 macrophages; Accuracy (%

327

Bias) and precision (% R.S.D.) data for the standards, namely rosmarinic acid (1)

328

and methyl rosmarinate (3); Supplemental figures: The fractionation procedure of S.

329

glabra and the effect of the various different fractions on NF-κB activity in

330

LPS-stimulated macrophages; Typical HPLC chromatograms for the n-BuOH

331

extract of S. glabra, and for the standards rosmarinic acid and methyl rosmarinate.

332

The n-BuOH fraction and a mixture of compounds 1 and 3 (22:1) inhibit

333

pro-inflammatory cytokine production in LPS-induced RAW 264.7 macrophages;

334

The effect of the n-BuOH fraction and a mixture of compounds 1 and 3 (22:1) on

335

ROS production in LPS-induced RAW 264.7 macrophages; Supplemental

336

experiment: Quantitative analysis of rosmarinic acid and methyl rosmarinate. These

337

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

338

Funding sources

19



339

This study was supported by two research grants (MOST 105-2320-B-077-003 and

340

MOST 103-2320-B-010-007-MY3) from the Ministry of Science and Technology,

341

Taiwan, ROC.

342 343

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antioxidant and antiviral activities. Food Chem. 2014, 152, 300-306.

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41. Li, W. Y.; Chiu, L. C. M.; Lam, W. S.; Wong, W. Y.; Chan, Y. T.; Ho, Y. P.;

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Bax/Bcl-2 ratio. Oncol. Rep. 2007, 17, 425-431.

473 474

27



475

Figure captions

476

Figure 1. Methyl isorinate inhibits NF-κB activity in LPS-stimulated

477

macrophages

478

(A) The structures of methyl isorinate (designated as compd. 2) (B) The

479

RAW264.7/Luc-P1 macrophages (1.5x105 cells in MP-24 plates) were treated with

480

compd. 2 or 0.1% DMSO for 1 h, followed by LPS treatment (10 ng/mL) for 24 h.

481

The luciferase activity of the treated groups was measured. (C) RAW264.7

482

macrophages (1.5x106 cells in MP-6 plates) were pre-treated with compd. 2 or 0.1%

483

DMSO for 2 h, followed by LPS treatment (1 µg/mL) for 30 min. IκB

484

phosphorylation and β-actin expression were detected by Western blotting. β-actin

485

served as the loading control. (D) RAW264.7 macrophages (1x104 cells in MP-96

486

plates) were treated with compd. 2 or 0.1% DMSO for 24 h, then the viability of

487

treated cells was measured using the MTT assay. Andro (andrographolide) was used

488

as the positive control. * indicates significant difference versus the LPS-treated

489

vehicle control (p < 0.05).

490 491

Figure 2. Methyl isorinate inhibits pro-inflammatory molecule production in

492

LPS-induced RAW 264.7 macrophages.

493

(A) The RAW 264.7 macrophages (1.5x105 in MP-24 plates) were treated with 28


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494

compd. 2 or 0.1% DMSO for 1 h, followed by LPS (10 ng/mL) treatment for 24 h.

495

The amount of TNF-α and IL-6 present in the culture supernatants was then

496

measured using an appropriate ELISA assay. Andro (andrographolide) acted as the

497

positive control. An asterisk (*) indicates a significant difference versus the

498

LPS-treated vehicle control (p < 0.05). (B) RAW 264.7 macrophages (5x105 in

499

MP-6 plates) were treated with compd. 2 or vehicle (0.1% DMSO) for 1 h, followed

500

by LPS (50 ng/mL) treatment for 24 h. The expression of iNOS, COX-2 and β-actin

501

was measured by Western blotting. Andro (andrographolide) acted as the positive

502

control. An asterisk (*) indicates p < 0.05 versus LPS-treated vehicle group.

503 504

Figure 3. Methyl isorinate suppresses ROS production in LPS-induced

505

macrophages

506

RAW 264.7 macrophages (5x105 in MP-6 plates) were treated with compd. 2 or

507

vehicle for 1 h, followed by LPS (50 ng/mL) treatment for 24 h. The cells were

508

incubated with H2DCFDA and their fluorescence intensity measured using flow

509

cytometry. (A) The fluorescent cell population of all treated groups. (B)

510

Quantification data from three independent experiments are shown. Fisetin was used

511

as the positive control. An asterisk (*) indicates p < 0.05 versus the LPS-treated

512

vehicle group. 29



513 514

Figure 4. The anti-inflammatory effects of the rosmarinic acid analogs on

515

LPS-stimulated macrophages

516

(A) The structures of the rosmarinic acid analogs. (B) RAW264.7/ Luc-P1

517

macrophages (1.5x105 cells in MP-24 plates) were treated with the indicated

518

compounds (10 µM) or 0.1% DMSO for 1 h, followed by LPS treatment (10 ng/mL)

519

for 24 h. The luciferase activity of the treated groups was measured. In (C) and (D),

520

the RAW 264.7 macrophages (1.5x105 in MP-24 plates) were treated with the

521

indicated compounds (10 µM) or 0.1% DMSO for 1 h, followed by LPS (10 ng/mL)

522

treatment for 24 h. The amount of TNF-α and IL-6 present in the culture

523

supernatants was measured by an appropriate ELISA assay. Andro (andrographolide)

524

was used as the positive control. An asterisk (*) indicates a significant difference

525

versus the LPS-treated vehicle control (p < 0.05).

526 527

Figure 5. The anti-inflammatory effects of caffeic acid and its structural

528

analogs on LPS-stimulated macrophages

529

(A) The structures of the caffeic acid analogs. (B) The luciferase activities of caffeic

530

acid and its analogs (10 µM) in LPS-treated RAW 264.7 macrophages were

531

examined. The production of (C) TNF-α and (D) IL-6 in the culture medium of the 30


Page 30 of 40

Page 31 of 40


532

LPS-activated RAW 264.7 macrophages pretreated with the indicated compounds

533

(10 µM) is shown. The treatment protocols and experimental procedures are

534

identical to those described in Figure 4. Andro (andrographolide) was used as the

535


536

LPS-treated vehicle control (p < 0.05).

537 538

Figure 6. The anti-inflammatory effects of the shikimic acid analogs on LPS-

539

stimulated macrophages

540

(A) The structures of the shikimic acid analogs (B) The luciferase activities of

541

shikimic acid analogs (10 µM) in LPS-treated RAW 264.7 macrophages were

542

measured. The expression of (C) TNF-α and (D) IL-6 in the culture medium of

543

LPS-activated RAW 264.7 macrophages pretreated with the shikimic acid analogs

544

(10 µM) are shown. The treatment protocols and experimental procedures are

545

identical to those described in Figure 4. Andro (andrographolide) was used as the

546


547

LPS-treated vehicle control (p < 0.05).

548 549

Figure 7. The anti-inflammatory effects of the benzoic acid analogs on LPS-

550

stimulated macrophages 31



551

(A) The structures of the benzoic acid analogs. (B) The luciferase activities of the

552

benzoic acid analogs (10 µM) in LPS-treated RAW 264.7 macrophages were

553

measured. The secretion of (C) TNF-α and (D) IL-6 into the culture medium of the

554

LPS-treated RAW 264.7 macrophages preincubated with the benzoic acid analogs

555

(10 µM) was measured. The treatment protocol and experimental procedures are

556

identical to those in Figure 4. Andro (andrographolide) was used as the positive

557

control. An asterisk (*) indicates a significant difference versus the LPS-treated

558

vehicle control (p < 0.05).

32


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Figure 1

33



Figure 2

34


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Figure 3

35



Figure 4

36


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Figure 5

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Figure 6

38


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Figure 7

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337x254mm (72 x 72 DPI)


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Anti-inflammatory Principles from

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