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Metabolic Fate of Luteolin in Rats: Its Relation to Anti-inflammatory Effect Ayako Kure, Kiyotaka Nakagawa, Momoko Kondo, Shunji Kato, Fumiko Kimura, Akio Watanabe, Naoki Shoji, Sakiko Hatanaka, Tojiro Tsushida, and Teruo Miyazawa J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00964 • Publication Date (Web): 12 May 2016 Downloaded from http://pubs.acs.org on May 12, 2016

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Journal of Agricultural and Food Chemistry

Metabolic Fate of Luteolin in Rats: Its Relation to Anti-inflammatory Effect Ayako Kure,1 Kiyotaka Nakagawa,*,1 Momoko Kondo,1 Shunji Kato,1 Fumiko Kimura,1 Akio Watanabe,2 Naoki Shoji,3 Sakiko Hatanaka,4 Tojiro Tsushida,5 and Teruo Miyazawa,6,7

1

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science,

Tohoku University, Sendai, Miyagi 981-8555, Japan 2

Food Function Research Team, Saito Laboratory, Japan Food Research Laboratories,

Ibaraki, Osaka 567-0085, Japan 3

Miyagi Prefecture Watari Agricultural Promotion Center, Watari, Miyagi 989-2301,

Japan 4

Industrial Technology Institute, Miyagi Prefectural Government, Sendai, Miyagi

981-3206, Japan 5

Department of Food Management, School of Food, Agricultural and Environmental

Sciences, Miyagi University, Sendai, Miyagi 982-0215, Japan 6

Food and Biotechnology Innovation Project, New Industry Creation Hatchery Center

(NICHe), Tohoku University, Sendai, Miyagi 980-8579, Japan 7

Food and Health Science Research Unit, Graduate School of Agricultural Science,

Tohoku University, Sendai, Miyagi 981-8555, Japan

AUTHOR INFORMATION Corresponding Author *(K.N.) Phone: 81-22-717-8906. Fax: 81-22-717-8905. E-mail: [email protected]. 1

ACS Paragon Plus Environment


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1

ABSTRACT: Luteolin is a naturally occurring flavone that reportedly has

2

anti-inflammatory effects. Because most luteolin is conjugated following intestinal

3

absorption, free luteolin is likely present at low levels in the body. Therefore, luteolin

4

metabolites are presumably responsible for luteolin bioactivity. Here we confirmed that

5

luteolin glucuronide, especially luteolin-3’-O-glucuronide, is the major metabolite

6

found in plasma after oral administration of luteolin (aglycone) or luteolin glucoside

7

(luteolin-7-O-glucoside)

8

luteolin-7-O-glucuronide were also detectable together with luteolin-3’-O-glucuronide

9

in the liver, kidney and small intestine. Next, we prepared these luteolin glucuronides

10

and compared the anti-inflammatory effects of luteolin and luteolin glucuronides on

11

gene expression in lipopolysaccharide-treated RAW264.7 cells. Luteolin glucuronides,

12

especially luteolin-7-O-glucuronide, reduced expression of inflammatory genes in the

13

cells, although their effects were weaker than those of luteolin. These results indicate

14

that the active compound responsible for the anti-inflammatory effect of luteolin in vivo

15

would be luteolin glucuronide and/or residual luteolin.

16

KEYWORDS: luteolin, luteolin glucoside, luteolin glucuronide, metabolic fate,

17

anti-inflammatory

to

rats.

Luteolin-4’-O-glucuronide

18 2


and

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19

20

Luteolin (Figure 1) is a naturally occurring flavone and found in high concentrations in

21

bell pepper, celery and perilla.1-3 Luteolin generally exists as glucoside (e.g.,

22

luteolin-7-O-glucoside), and its concentration reaches 11 mg/kg in dried powder of bell

23

pepper.2 Luteolin and/or luteolin glucoside have been known to have anti-inflammatory,

24

anti-oxidative and anti-cancer activity in vivo.4-6 Among these properties, many

25

researches have focused on anti-inflammatory effect.7-9 For instance, it is reported that

26

mice receiving lipopolysaccharide (LPS, 32 mg/kg, intraperitoneally) exhibited high

27

mortality with only 4% of the animals surviving seven days after the LPS challenge, on

28

the contrary, mice that had received luteolin (0.2 mg/kg, intraperitoneally) before LPS

29

showed an increased survival rate with 48% remaining alive on day 7.4

INTRODUCTION

30

Despite of increasing number of the functional studies,4-9 researches about absorption

31

and metabolism of luteolin and luteolin glucoside have been limited. Lin et al.

32

investigated absorption of luteolin and luteolin-7-O-glucoside in rats.10 They reported

33

that luteolin is absorbed into rat body, whereas luteolin-7-O-glucoside is primarily

34

hydrolyzed to luteolin in the gastrointestinal tract and then absorbed into the systemic

35

circulation. Shimoi et al. reported that when luteolin is absorbed through intestine in rats,

36

most

of

the

luteolin

is

converted

to

luteolin

glucuronide.11,12

3


Similarly,


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37

luteolin-7-O-glucoside is hydrolyzed and converted to mainly luteolin glucuronide after

38

administration of luteolin-7-O-glucoside to rats. Unmetabolized luteolin and

39

luteolin-7-O-glucoside are found in blood at low levels. Thus, luteolin conjugates, but

40

not luteolin and luteolin glucoside, are presumed to be responsible for biological

41

activity inside the body.

42

In spite of the speculation, the bioactivities of luteolin conjugates have not been well

43

investigated, due to uncertainty in the position of glucuronide group in luteolin

44

glucuronide present in in vivo (Figure 1) and thereby difficulty in preparation of

45

authentic luteolin conjugates. Hence, in this study, after confirming that luteolin

46

glucuronide is the main metabolite of luteolin and luteolin-7-O-glucoside in rat, we

47

determined the position of glucuronide group by using high performance liquid

48

chromatography-tandem mass spectrometry (HPLC-MS/MS) and reference luteolin

49

glucuronides

50

luteolin-7-O-glucuronide) prepared. Then, we compared anti-inflammatory effects of

51

these luteolin-glucuronides on inflammatory in lipopolysaccharide (LPS)-treated

52

RAW264.7 cells with those of luteolin, in order to evaluate which luteolin glucuronide

53

can be a bioactive molecule inside the body.

(luteolin-3’-O-glucuronide,

luteolin-4’-O-glucuronide

54 4


and

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55 56

MATERIALS AND METHODS Reagents. Luteolin was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo,

57

Japan).

Luteolin-7-O-glucoside

was

prepared

by

partial

hydrolysis

of

58

luteolin-7-O-glucosyl derivatives obtained from sweet pepper leaves.13,14 LPS

59

(Escherichia coli 0111) was obtained from Sigma (St. Louis, MO, USA). All other

60

chemicals and reagents used were of analytical grade or higher.

61

Preparation of luteolin glucuronide. Luteolin-3’-O-glucuronide was isolated from

62

50 g of dry rosemary leaves as reported by Borrás-Linares et al.15 Based on the previous

63

method for synthesis of quercetin-4’-O-glucuronide,16 luteolin-4’-O-glucuronide was

64

synthesized by reacting luteolin with acetobromo-α-D-glucuronic acid methyl ester.

65

Luteolin-7-O-glucuronide was isolated from 50 g of dry thyme leaves as reported by

66

Dapkevicius et al.17

67

Animal study. Male Sprague-Dawley rats (8 weeks of age; mean body weight 244 ±

68

4 g) were obtained from CLEA Japan, Inc. (Tokyo, Japan), and housed in cages at 23

69

°C with a 12 h light:dark cycle. The rats were acclimated with commercial rodent chow

70

(CE-2; CLEA Japan Inc.) and water for 1 week. After acclimatization, rats (n = 4-5)

71

were fasted for 12 h and then received either luteolin (20 mg/kg) or

72

luteolin-7-O-glucoside (20 mg/kg) by oral gavage using 1% sodium cholate as a 5



73

vehicle. Blood (about 0.3 mL) was collected into heparinized tubes from the tail vein

74

using a capillary tube at 0, 1, 3, 6, 12 and 24 h after the oral administration. The blood

75

was centrifuged at 1,000g for 15 min at 4 °C to prepare plasma. In a separate study,

76

livers, kidneys and small intestines (n = 3) were excised 6 h after oral administration of

77

luteolin (20 mg/kg) or luteolin-7-O-glucoside (20 mg/kg). Plasma and organ samples

78

were stored at -80 °C until further analysis. These protocols were reviewed by the

79

Committee on the Ethics of Animal Experiments and carried out in accordance with the

80

Animal Experiment Guidelines of Tohoku University (Sendai, Japan). The permit

81

number for this animal experiment is 2015-Noudou-036.

82

Extraction. Plasma (100 µL) was mixed with 300 µL acetonitrile and centrifuged at

83

1,000g for 10 min at 4 °C. The supernatant was then collected from the precipitate by

84

decantation. Methanol (300 µL) was added to the precipitate, mixed, and centrifuged at

85

1,000g for 10 min at 4 °C. The resulting supernatants were collected, combined, dried,

86

and redissolved in 10% acetonitrile aqueous solution. Liver, kidney and small intestine

87

tissues were homogenized with saline containing 1 mM ethylenediaminetetraacetic acid

88

(EDTA) to prepare a 30% (w/v) homogenate solution. The homogenate (500 µL) was

89

extracted with acetonitrile (1.5 mL) and methanol (1.5 mL) in the same way as plasma.

90

The resultant extract was filtered with a Chromatodisc 13P (0.45 µm pore size, GL 6


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91

Science, Tokyo, Japan) filter, and a final aliquot of 10 µL was subjected to

92

HPLC-MS/MS analysis.

93

HPLC-MS/MS. For HPLC-MS/MS, a C18 column (CAPCELL PAK C18 MGII S3,

94

4.6 × 150 mm; Shiseido) was used at 40 °C. The mobile phase consisted of two

95

components: A, water containing 0.1% trifluoroacetic acid and B, acetonitrile. The

96

gradient profile was as follows: 0-20 min, 90-70% A linear; 20-25 min, 70-50% A

97

linear. The flow rate was 0.8 mL/min. The mobile phase was split at the postcolumn.

98

One of the split eluents (flow rate = 0.2 mL/min) was sent to a 4000 QTRAP

99

HPLC-MS/MS (AB SCIEX, CA, USA), while the other (flow rate = 0.6 mL/min) was

100

discarded. MS/MS parameters of luteolin-3’-O-glucuronide, luteolin-4’-O-glucuronide,

101

luteolin-7-O-glucuronide and luteolin were optimized with their standards under

102

electrospray ionization (negative). The parameters were as follows: turbo gas

103

temperature, 600 °C; spray voltage, -4500 V; nebulizer gas, 40 psi; auxiliary gas, 40 psi;

104

curtain

105

lute-olin-4’-O-glucuronide, luteolin-7-O-glucuronide and luteolin were detected by

106

using multiple-reaction monitoring (MRM) for the transition of precursor ions to

107

product ions: luteolin-3’-O-glucuronide, m/z 461 > 285 (collision energy (CE), -32 V;

108

decluttering potential (DP), -95 V); luteolin-4’-O-glucuronide, m/z 461 > 285 (CE, -32

gas,

20

psi;

collision

gas,

4.0.

7


Luteolin-3’-O-glucuronide,


109

V; DP, -95 V); luteolin-7-O-glucuronide, m/z 461 > 285 (CE, -32 V; DP, -95 V);

110

luteolin, m/z 284 > 133 (CE, -48 V; DP, -100 V). MS/MS detection limits of luteolin

111

glucuronide and luteolin were in the range of 0.1 to 0.2 pmol at a signal-to-noise ratio of

112

3. Their concentrations (e.g., luteolin-3’-O-glucuronide) in the sample extracts were

113

calculated from respective standard curves. Extraction efficacy of 10 pmol

114

luteolin-3’-O-glucuronide standard spiked with rat plasma (100 µL) was 70%. Other

115

flavonoids were analyzed using MRM transitions defined in the litera-ture18-20 or

116

predicted MRM ion pairs (CE, -50 V; DP, -90 V): luteolin glucoside, m/z 447 > 285;

117

luteolin sulphate, m/z 365 > 285; luteolin diglucuronide, m/z 637 > 285; luteolin

118

glucoside glucuronide, m/z 632 > 285; luteolin glucoside sulphate, m/z 637 > 285;

119

luteolin glucuronide sulphate, m/z 541 > 285; methylated luteolin, m/z 301 > 285;

120

methylated luteolin glucuronide, m/z 477 > 285.

121

Cells. Mouse macrophage RAW264.7 cells were obtained from the RIKEN

122

BioResource Center (Tukuba, Japan) and cultured in Eagle's minimum essential

123

medium (EMEM, Sigma) supplemented with 10% fetal bovine serum (FBS, Dainippon

124

Sumitomo Pharmaceutical, Osaka, Japan), 1 mM sodium pyruvate, 0.1 mM

125

non-essential amino acids, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C

126

with 5% CO2. 8


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127

Preparation

of

test

medium.

Test

samples


128

luteolin-4’-O-glucuronide, luteolin-7-O-glucuronide and luteolin) were dissolved in

129

dimethyl sulfoxide (DMSO) at a concentration of 50 mM. The stock solution was

130

diluted with test medium (EMEM containing 10% FBS) to achieve the desired final

131

concentration (e.g., 0-50 µM luteolin-3’-O-glucuronide). The final concentration of

132

DMSO in the test medium was less than 0.1% (v/v), which did not affect cell viability.

133

Medium with solvent (DMSO) alone was similarly prepared and used as the control

134

medium.

135

Cell cytotoxicity assays. RAW264.7 cells (1.0 × 105) were pre-incubated with 10%

136

FBS/EMEM in 96-well plates. Twenty-four hours later, the medium was replaced with

137

test medium containing 0-50 µM luteolin-3’-O-glucuronide, luteolin-4’-O-glucuronide,

138

luteolin-7-O-glucuronide or luteolin for 24 h. Then, the number of viable cells was

139

evaluated using WST-1 reagent (Dojindo Laboratories, Kumamoto, Japan) according to

140

the manufacturer's instructions. In brief, 10 µL WST-1 reagent was added to the

141

medium and incubated at 37 °C for 1 h. Absorbance (450/655 nm) of the medium was

142

measured with a microplate reader.

143

Sample treatment and total RNA extraction. After a 24 h pre-incubation of

144

RAW264.7 cells (1.0 × 106) with 10% FBS/EMEM in a 5 cm dish, the cells were 9



145

treated with the test medium for 24 h. LPS was then added to the test medium at a final

146

concentration of 1 µg/mL. After further incubation for 3 h, total RNA was extracted

147

from the cells using an RNeasy Mini Kit (Qiagen, Tokyo, Japan). Similarly, total RNA

148

was extracted from cells that were not treated with flavonoid and LPS.

149

DNA microarray and real time RT-PCR analysis. In order to identify genes

150

related to the anti-inflammatory effect of luteolin, RAW264.7 cells were treated with or

151

without luteolin in the presence of LPS, and total RNA was extracted as described

152

above. A DNA microarray was then performed using the allergy chip Genopal

153

(Mitsubishi Rayon, Tokyo, Japan) according to the manufacturer’s protocol. After

154

selecting genes from the DNA microarray, real time RT-PCR analysis was performed.

155

In brief, RAW264.7 cells were treated with or without luteolin-3’-O-glucuronide,

156

luteolin-4’-O-glucuronide, luteolin-7-O-glucuronide or luteolin in the presence and

157

absence of LPS. cDNA was then synthesized from total RNA (500 ng) using

158

PrimeScript Master mix (Takara Bio, Otsu, Japan), and PCR amplification was

159

performed using a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories,

160

New South Wales, Australia) with SYBR Premix Ex Taq II (Takara Bio, Otsu, Japan)

161

and gene-specific primers for interleukin-6 (IL-6), interleukin-1 beta (IL-1β), nuclear

162

factor of kappa light polypeptide gene enhancer in B-cells 1 (NF-κB1), CC chemokine 10


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163

ligand 2 (Ccl2), CC chemokine ligand 3 (Ccl3), CC chemokine ligand 5 (Ccl5), Jun B

164

Proto-Oncogene (JunB), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

165

(Table 1). PCR conditions were 95 °C for 3 min, followed by 40 cycles of 95 °C for 10

166

s and 55 °C for 30 s.

167

Statistical analysis. Data are expressed as means ± standard error (SE). One-Way

168

ANOVA followed by Tukey’s test was per-formed for multiple comparisons.

169

Differences were considered significant at P < 0.05.

170 171

RESULTS AND DISCUSSION

172

Preliminary single oral supplementation study of luteolin. Unlike the flavonoids

173

catechin and quercetin, studies concerning the metabolic fate of luteolin are limited. As

174

described by the Shimoi group,11,12 there are some studies on the bioavailability of

175

luteolin after administration of C. morifolium flower extract that showed the luteolin in

176

the extract was absorbed by rats and humans.21,22 However, in their analyses, because

177

they investigated samples (e.g., plasma and urine) cleaved into aglycone, it remained

178

unknown whether the circulated luteolin exists as a free form or any conjugated form.

179

Shimoi and her coworkers reported that after administration of C. morifolium flower

180

extract to rats, luteolin and luteolin glucoside (luteolin-7-O-glucoside) were quickly 11



181

absorbed and were present mainly as luteolin glucuronide in plasma.11,12 Thus, the

182

results from this and other studies11,12,21,22 suggest that luteolin glucuronide is

183

responsible for in vivo biological activity, although the position of the glucuronide

184

group in luteolin glucuronide remained unclear. Identification of this position is

185

important given that the glucuronide group position in flavonoids is generally

186

considered to be closely related to biological function.23 On the other hand,

187

HPLC-MS/MS can be used to analyze biomolecules, and offers advantages over

188

conventional technics (e.g., HPLC-UV). HPLC-MS/MS provides neutral loss scanning,

189

precursor ion scanning and MRM, which are useful for structural identification of the

190

analytes present in vivo.20 Recently, Shimoi and her coworkers identified luteolin

191

glucuronide together with luteolin and luteolin glucoside, from plasma of rats

192

supplemented with C. morifolium flower extract using HPLC-MS/MS with MRM.12

193

From the above reasons, we first performed preliminary single oral supplementation

194

study of luteolin (20 mg/kg) to rats, in order to analyze luteolin glucuronide and other

195

metabolites that might possibly be present in plasma, such as luteolin glucoside, luteolin

196

sulphate, luteolin diglucuronide, luteolin glucoside glucuronide, luteolin glucoside

197

sulphate, luteolin glucuronide sulphate, methylated luteolin and methylated luteolin

198

glucuronide, by using HPLC-MS/MS with MRM. The preliminary experiment clearly 12


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199

showed that luteolin glucuronide is the main metabolite of luteolin in rats 6 h after

200

administration (data not shown). Luteolin diglucuronide and luteolin glucuronide

201

sulphate were also found in plasma, while unmetabolized luteolin was seen at low levels.

202

Luteolin glucoside, luteolin sulphate, luteolin glucoside glucuronide, luteolin glucoside

203

sulphate, methylated luteolin and methylated luteolin glucuronide were trace or not

204

detected. Based on these results, we focused on luteolin glucuronide and tried to

205

determine the position of the glucuronide group.

206

Preparation and HPLC-MS/MS analysis of reference luteolin glucuronides. Prior

207

to identifying the position of the glucuronide group in luteolin glucuronide, we prepared

208

several

209


210

luteolin-3’-O-glucuronide and luteolin-7-O-glucuronide were extracted from rosemary

211

and

212


213

acetobromo-α-D-glucuronic acid methyl ester.16 The purity of each prepared

214

glucuronide was > 95% as evaluated by HPLC. These glucuronides were used as

215

reference standards.

thyme

reference

leaves,

luteolin and

respectively, was

glucuronides:

luteolin-3’-O-glucuronide,

luteolin-7-O-glucuronide.

In

this

and then chromato-graphically synthesized

by

reacting

13


study,

purified.15,17

luteolin

with


Page 14 of 37

216

Using the purified reference standards, we then optimized analytical conditions.

217

Analysis of standard luteolin-3’-O-glucuronide by MS/MS with flow injection showed

218

an intense molecular ion at m/z 461. Product ion scanning was conducted for this ion,

219

and

220


221

fragmentation pattern (Figure 2B,C). The identified ions (m/z 285) allowed for detection

222

of analytes by HPLC-MS/MS with MRM. In the MRM chromatogram (Figure 2D),

223

luteolin-3’-O-glucuronide, luteolin-4’-O-glucuronide and luteolin-7-O-glucuronide

224

standards were clearly detected at 19 min, 18 min and 15 min, respectively. Notably, the

225

CAPCELL PAK C18 MGII S3 column was very effective for glucuronide separation.

226

Similarly, HPLC-MS/MS conditions of luteolin were optimized, and luteolin was

227

detected at 23 min (Figure 2D). Standard curves were linear for each analyte in the

228

concentration range from 1 to 20 pmol. The sensitivity and selectivity of this

229

HPLC-MS/MS with MRM method indicates its potential application as a tool for

230

evaluating luteolin bioavailability.

fragment

ions

(e.g., and

m/z

285)

were

identified

luteolin-7-O-glucuronide

(Figure

showed

the

2A). same

231

Luteolin and luteolin-7-O-glucoside are absorbed and exist mainly as

232

luteolin-3’-O-glucuronide in rat plasma. We next performed a single oral

233

supplementation study of luteolin and luteo-lin-7-O-glucoside. Rats were divided into 2 14


Page 15 of 37


234

groups, and either 20 mg/kg luteolin or 20 mg/kg luteolin-7-O-glucoside was

235

administered orally. Six hours after the administration representative MRM

236

chromatograms of rat plasma extracts from animals receiving either luteolin and

237

luteolin-7-O-glucoside showed a large peak of luteolin-3’-O-glucuronide together with

238

a very small peak for luteolin (Figure 3A). The HPLC-MS/MS with MRM detection of

239

luteolin and its metabolites was reproducible and was not affected when plasma extract

240

samples were stored at -30 °C for 1 month. For rats administered with luteolin,

241

time-dependent changes (0, 1, 3, 6, 12 and 24 h) showed that plasma

242

luteolin-3’-O-glucuronide concentrations increased and reached a maximum level at 3 h

243

(Figure

244

luteolin-7-O-glucoside-administered group, maximum time and concentration of plasma

245

luteolin-3’-O-glucuronide were longer and somewhat lower, respectively, relative to

246

animals

247

luteolin-3’-O-glucuronid concentrations were roughly comparable to those of previous

248

studies (~14 µM).11,12 In addition to luteolin-3’-O-glucuronide, substantial amounts of

249

luteolin-4’-O-glucuronide and luteolin-7-O-glucuronide were also detected in the liver,

250

kidney and small intestine at 6 h after oral administration of luteolin (20 mg/kg) and

251

luteolin-7-O-glucoside (20 mg/kg) (Figure 3A). These concentrations were shown in

3B,

that

Table

received

S1).

After

luteolin

that,

(Figure

the

3B,

levels

Table

15


decreased.

S1).

These

In

the

plasma


Page 16 of 37

252

Table S2. Further studies about the tissue determination including extraction recovery

253

are needed, because we could not check the recovery from tissues due to limitations of

254

amounts of luteolin glucuronide standards. Nevertheless, to the best of our knowledge,

255

this is the first study to show the profile of luteolin metabolites in tissues. These results

256

indicate that luteolin and luteolin glucoside (luteolin-7-O-glucoside) are absorbed and

257

mainly exist as luteolin-3’-O-glucuronide in plasma. Since luteolin-4’-O-glucuronide

258

and luteolin-7-O-glucuronide were also present in tissues, these luteolin-glucuronides

259

would likely have in vivo bioactivity. As a point of reference, luteolin glucuronide is

260

thought to be formed by phase II enzymes in small intestine, liver, and kidney.24

261

However, why luteolin glucuronides such as luteolin-3’-O-glucuronide, but not sulfate

262

and methyl modifications, are preferentially formed in vivo is unclear, and requires

263

additional

264

luteolin-7-O-glucoside, the glucoside is suggested to be hydrolyzed to luteolin by

265

bacteria on the surface of the intestinal mucosa.10 This may be related to our findings

266

that

267

luteolin-7-O-glucoside-administered group was longer than for luteolin-administered

268

group.

investigation.

maximum

time

With

of

regard

plasma

to

the

hydrolysis


16


mechanism

(Figure

3B)

of

for

Page 17 of 37


269

Luteolin glucuronide inhibits LPS-induced inflammatory gene expression. As

270

mentioned above, the anti-inflammatory effects of luteolin have recently attracted

271

attention,4,7-9 and many in vitro studies showed the potent anti-inflammatory activity of

272

luteolin.25-29 For instance, Park et al.29 reported that luteolin could reduce inflammatory

273

responses in LPS-activated RAW264.7 cells by attenuating the activation of

274

transcription

275

luteolin-7-O-glucoside alone impeded NF-κB activation,29 which may imply that

276

structural modifications of luteolin (e.g., glycosylation and glucuronidation) reduce its

277

bioactivity. Given that in rats luteolin is present as luteolin glucuronide rather than

278

luteolin itself or other metabolites (Figure 3), we felt that determining the bioactivity of

279

luteolin glucuronide, which has not been thoroughly investigated, would be of primary

280

importance.

281

luteolin-3’-O-glucuronide, luteolin-4’-O-glucuronide and luteolin-7-O-glucuronide in

282

LPS-treated RAW264.7 cells with those of luteolin.

283

factors

As

(NF-κB

such,

we

and

AP-1).

compared

These

the

authors

also

anti-inflammatory

found

effects

that

of

To determine the optimal dose, we first treated RAW264.7 cells with 0-50 µM

284




285

luteolin in the absence of LPS. After incubation for 24 h, the number of viable cells was

286

determined using a WST-1 assay. Luteolin glucuronides (0-50 µM) either had no effect 17


or


287

or slightly increased cell viability (Figure 4). In contrast, cell viability was suppressed to

288

~65% following 25 µM luteolin treatment, which may be due to luteolin-induced

289

apoptosis.30 We chose the 25 µM concentration to compare the anti-inflammatory

290

effects of luteolin glucuronide and luteolin. In this regard, the concentration of 25 µM

291

was above the peak plasma luteolin-3’-O-glucuronide concentration (4.1 µM) (Figure

292

3B, Table S1). However, we think that the level in our in vitro study (25 µM) represents

293

a physiologically attainable concentration, especially in a local site in vivo, considering

294

the results of previous reports showing that the peak plasma concentrations of ~14 µM

295

were achieved in rats after administration of 3 mg luteolin.11 In addition, many in vitro

296

studies tested 10-40 µM luteolin and reported that 20~ µM luteolin showed effects.5,31,32

297

Taken together, in the present in vitro study, we compared effects of 25 µM luteolin

298

with those of luteolin glucuronides.

299

We

treated

RAW264.7

cells

with

25

µM


300

luteolin-4’-O-glucuronide, luteolin-7-O-glucuronide or luteolin for 24 h before LPS (1

301

µg/mL) was added. Further incubation was performed for 3 h, and mRNA expression of

302

genes related to inflammation (IL-6, IL-1β, NF-κB1, Ccl2, Ccl3, Ccl5 and JunB) was

303

evaluated by real time RT-PCR. These genes were chosen because we confirmed that

304

luteolin affected their expression in a preliminary DNA microarray analysis (data not 18


Page 18 of 37

Page 19 of 37


305

shown). Similar effects of luteolin on these genes were also reported in previous

306

studies.25-29 PCR analysis clearly revealed that LPS induced notable expression of IL-6,

307

IL-1β, NF-κB1, Ccl2, Ccl3, Ccl5 and JunB, and luteolin glucuronide treatment reduced

308

these expression levels (Figure 5). This effect was weaker than that for luteolin itself.

309

These results suggested that the anti-inflammatory activity of luteolin is (to some

310

extent) maintained after conversion of luteolin to glucuronide. The inhibitory effect of

311

glucuronides could be ranked as luteolin-7-O-glucuronide > luteolin-4’-O-glucuronide

312

>

313

luteolin-3’-O-glucuronide and luteolin-4’-O-glucuronide, has a catechol structure,

314

which may be related to the potent inhibitory effect of luteolin-7-O-glucuronide.

315


In

conclusion,

we

(Figure

showed

that

5).

after

Luteolin-7-O-glucuronide,

administration

of

but

luteolin

not

and

316

luteolin-7-O-glucoside to rats, these molecules were absorbed and existed mainly as

317

luteolin glucuronide (e.g., luteolin-3’-O-glucuronide) in the body. Luteolin glucuronide,

318

especially luteolin-7-O-glucuronide, could reduce the expression of inflammatory genes

319

in LPS-treated RAW264.7 cells. For example, luteolin-7-O-glucuronide showed

320

45-74% inhibition of LPS-induced expression of IL-6, IL-1β, NF-κB1, Ccl2, Ccl3 and

321

Ccl5 in the cells. It is therefore likely that the active molecule responsible for the

322

anti-inflammatory activity in vivo would be luteolin glucuronide and/or residual luteolin 19



323

present in the body. Meanwhile, β-glucuronidase released from neutrophils at the site of

324

inflammation may hydrolyze luteolin glucuronide to luteolin that in turn attenuates

325

inflammation.33 Further in vitro and in vivo investigations will be required to further

326

understand the absorption, metabolism and effects of luteolin and luteolin glucoside in

327

vivo.

328 329

330

We thank Natsumi Hayasaka (Food and Biodynamic Chemistry Laboratory, Graduate

331

School of Agricultural Science at Tohoku University) for her assistance in the

332

preparation of the manuscript.

ACKNOWLEDGMENT

333 334

335

Supporting Information

336

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

337

website.

338

Plasma and tissue concentrations after oral administration of luteolin (20 mg/kg) or

339

luteolin-7-O-glucoside (20 mg/kg).

ASSOCIATED CONTENT

340 20


Page 20 of 37

Page 21 of 37


341

342

(1) Miean, K.H.; Mohamed, S. Flavonoid (myricetin, quercetin, kaempferol, luteolin,

343

and apigenin) content of edible tropical plants. J. Agric. Food Chem. 2001, 49, 106-112.

344

(2) Sun, T.; Xu, Z.; Wu, C.T.; Janes, M.; Prinyawiwatkul, W.; No, H.K. Antioxidant

345

activities of different colored sweet bell peppers (Capsicum annuum L.). J. Food Sci.

346

2007, 72, 98-102.

347

(3) Jeon, I.H.; Kim, H.S.; Kang, H.J.; Lee, H.S.; Jeong, S.I.; Kim, S.J.; Jang, S.I.

348

Anti-inflammatory and antipruritic effects of luteolin from perilla (P. frutescens L.)

349

leaves. Molecules 2014, 19, 6941-6951.

350

(4) Kotanidou, A.; Xagorari, A.; Bagli, E.; Kitsanta, P.; Fotsis, T.; Papapetropoulos, A.;

351

Roussos, C.; Theodore, F. Luteolin reduces lipopolysaccharide-induced lethal toxicity

352

and expression of proinflammatory molecules in mice. Am. J. Respir. Criti. Care Med.

353

2002, 165, 818-823.

354

(5) Androutsopoulos, V.P.; Spandidos, D.A. The flavonoids diosmetin and luteolin

355

exert

356

CYP1A-catalyzed metabolism, activation of JNK and ERK and P53/P21 up-regulation.

357

J. Nutr. Biochem. 2013, 24, 496-504.

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inflammation-associated gene expression by blocking NF-kappaB and AP-1 activation

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pathway in mouse alveolar macro-phages. Life Sci. 2007, 81, 23-24.

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(27) Funakoshi-Tago, M.; Nakamura, K.; Tago, K.; Mashino, T.; Kasahara, T.

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Anti-inflammatory activity of structurally related flavonoids, apigenin, luteolin and

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fisetin. Int. Immunopharmacol. 2011, 11, 1150-1159.

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(28) Yang, H.; Liu, Q.; Ahn, J.H.; Kim, S.B.; Kim, Y.C.; Sung, S.H.; Hwang, B.Y.; Lee,

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M.K. Luteolin downregulates IL-1β-induced MMP-9 and -13 expressions in osteoblasts

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via inhibition of ERK signaling pathway. J. Enzyme Inhib. Med. Chem. 2012, 27,

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lipopolysaccharide-induced

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NF-κB/AP-1/PI3K-Akt signaling cascades in RAW 264.7 cells. Nutr. Res. Pract. 2013,

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7, 423-429.

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(30) Xu, T.; Li, D.; Jiang, D. Targeting cell signaling and apoptotic pathways by

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luteolin: cardioprotective role in rat cardiomyocytes following ischemia/reperfusion.

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C.M.;

Song,

Y.S.

Luteolin

inflammatory

and

luteolin-7-O-glucoside

responses

through

26


inhibit

modulation

of

Page 27 of 37


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(31) Sulaiman, G.M. In vitro study of molecular structure and cytotoxicity effect of

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luteolin in the human colon carcinoma cells. Eur. Food Res. Technol. 2015, 24, 83-90.

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(32) Ou, Y.C.; Li, J.R.; Kuan, Y.H.; Raung, S.L.; Wang. C.C.; Hung, Y.Y.; Pan, P.H.;

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Lu, H.C.; Chen, C.J. Luteolin sensitizes human 786-O renal cell carcinoma cells to

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TRAIL-induced apoptosis. Life Sci. 2014, 100, 110-117.

449

(33) Shimoi, K.; Nakayama, T. Glucuronidase deconjugation in inflammation. Methods

450

Enzymol. 2005, 400, 263-272.

451 452

Notes

453

The authors declare no competing ﬁnancial interest.

454 455

Funding

456

This study was supported in part by project (a scheme to revitalize agriculture and

457

fisheries in disaster area through deploying highly advanced technology), Ministry of

458

Agriculture, Forestry and Fisheries (MAFF), Japan.

27



Page 28 of 37

459

FIGURE CAPTIONS

460

Figure

461

(luteolin-7-O-glucoside)

462

luteolin-4’-O-glucuronide and luteolin-7-O-glucuronide).

463

Figure 2. Product ion mass spectra of standard luteolin-3’-O-glucuronide (m/z 461

464

[M-H]-)

465

luteolin-7-O-glucuronide (m/z 461 [M-H]-) (C) under electrospray ionization (negative).

466

Standard

467

luteolin-7-O-glucuronide (1 µM in 10% acetonitrile aqueous solution) was infused

468

directly into the MS/MS apparatus at a flow rate of 10 µL/min. Standard

469

luteolin-3’-O-glucuronide, luteolin-4’-O-glucuronide, luteolin-7-O-glucuronide and

470

luteolin (each 1 pmol) were analyzed by HPLC-MS/MS with MRM using m/z 461/285,

471

m/z 461/285, m/z 461/285 and m/z 284 > 133, respectively (D). Detailed analytical

472

conditions are described in the Materials and Methods.

473

Figure 3. Representative MRM chromatograms of luteolin-3’-O-glucuronide,

474

luteolin-4’-O-glucuronide, luteolin-7-O-glucuronide and luteolin in rat plasma and

475

tissues (liver, kidney and small intestine) at 6 h after oral administration of luteolin (20

476

mg/kg) (A). A similar chromatogram was obtained for luteolin-7-O-glucoside (20

1.

Chemical

(A),

structures and

of

luteolin

luteolin

aglycone,

glucuronide



(m/z

luteolin

glucoside


461

[M-H]-)

(B)


28


and

or

Page 29 of 37


477

mg/kg) administration. Time-dependent changes (0, 1, 3, 6, 12 and 24 h) in plasma

478

luteolin-3’-O-glucuronide concentration are shown (B). Values are means ± SE (n =

479

4-5). Detailed analytical conditions are described in Materials and Methods.

480

Figure

481

luteolin-7-O-glucuronide and luteolin on RAW264.7 cell viability. RAW264.7 cells (1.0

482

× 105) were pre-incubated with 10% FBS/EMEM in 96-well plates for 24 h. After

483

additional 24 h incubation of the cells with test medium containing 0-50 µM

484


485

luteolin, the number of viable cells was evaluated using the WST-1 method. Detailed

486

analytical conditions are described in the Materials and Methods. Values are means ±

487

SE (n = 6). Means without a common letter differ, P < 0.05.

488

Figure

489

luteolin-7-O-glucuronide and luteolin on mRNA expression of genes related to

490

inflammation in LPS-treated RAW264.7 cells. RAW264.7 cells (1.0 × 106) were

491

pre-incubated with 10% FBS/EMEM in a 5 cm dish for 24 h. The cells were then

492

treated

493

luteolin-7-O-glucuronide or luteolin for 24 h. Further incubation was performed in the

494

presence and absence of LPS (1 µg/mL) for 3 h, and mRNA expression of genes (IL-6,

4.

5.

with

Effects

Effect

25

of



of

µM





29


or




495

IL-1β, NF-κB1, Ccl2, Ccl3, Ccl5, JunB, and GAPDH) was evaluated by real time

496

RT-PCR. Detailed analytical conditions are de-scribed in Materials and Methods.

497

Values are means ± SE (n = 3). Means without a common letter differ, P < 0.05.

30


Page 30 of 37

Page 31 of 37


Table 1. Primer Pairs Used for RT-PCR Gene

GenBank ID

Primer sequence (5’ to 3’) Forward

Reverse

IL-6

NM_031168

GAGGATACCACTCCCAACAGACC

AAGTGCATCATCGTTGTTCATACA

IL-1β

NM_008361

TCCAGGATGAGGACATGAGCAC

GAACGTCACACACCAGCAGGTTA

NF-κB1

NM_008689

CAGCTCTTCTCAAAGCAGCA

TCCAGGTCATAGAGAGGCTCA

Ccl2

NM_011333

AGGTCCCTGTCATGCTTCTGG

CTGCTGCTGGTGATCCTCTTG

Ccl3

NM_011337

GAAGATTCCACGCCAATTCATC

GATCTGCCGGTTTCTCTTAGTC

Ccl5

NM_013653

GCTGCTTTGCCTACCTCTCC

TCGAGTGACAAACACGACTGC

JunB

NM_008416

TCCAGCGTATTTTGTATGTT

CTTCTCCCTCCTGTTAAATAC

GAPDH

NM_008084

CATGTTCCAGTATGACTCCACTC

GGCCTCACCCCATTTGATGT

31



Figure 1

Luteolin


Luteolin-7-O-glucoside


32


Luteolin-7-O-glucuronide

Page 32 of 37

Page 33 of 37


Figure 2

A

B 100

100

285.2

285.0

OH

Glucuronate O

HO

O

m/z 285

Relative intensity (%)


m/z 285 Luteolin-3’-O-glucuronide 461.2 133.0

0

OH

O


132.8

461.0

0 100

200

300 m/z

400

500

100

C

200

300 m/z

400

500

D 100

100 285.0

Luteolin-3’-O-glucuronide (461/285) Luteolin-4’-O-glucuronide (461/285) Luteolin (285/133)



m/z 285 Luteolin-7-O-glucuronide 461.0

Luteolin-7-O-glucuronide (461/285)

132.8

0

0 100

200

300 m/z

400

0

500

10

20 Time (min)

33


30


Page 34 of 37

Figure 3

A

100


Plasma

Luteolin (20 mg/kg) administration

Luteolin-3’-O-glucuronide Luteolin-4’-O-glucuronide

Luteolin

Luteolin-7-O-glucuronide 0 0

10

20

30

Time (min) 100



Liver


Luteolin-4’-O-glucuronide Luteolin

Luteolin-7-O-glucuronide 0 0

10

20

30

Time (min) 100


Kidney


Luteolin-3’-O-glucuronide Luteolin-4’-O-glucuronide Luteolin-7-O-glucuronide

Luteolin

0 0

10

20

30

Time (min) 100


Small intestine


Luteolin-4’-O-glucuronide Luteolin-7-O-glucuronide

Luteolin

Plasma luteolin-3’O-glcuronide (µM)

8

4000


6

3000

Luteolin-7-O-glucoside (20 mg/kg) administration

4

2000

2

1000

0

Plasma luteolin-3’O-glcuronide (µg/L)

B


0

0 1

3

6

12 Time (h)

24

34


Page 35 of 37


Figure 4

a

a

a

100

50

0

5

10

25

50

Cell viability (% of control)

a a

a

100

50

0

0

5

10

25

200 b b

50

a a

50

0

b

0

5

10

25

b

25

50

100

a

100

ab

a

Luteolin-4’-O-glucuronide concentration (µM)

Luteolin-3’-O-glucuronide concentration (µM) 120

a


a

0

a

140

a



160

b

50

Luteolin concentration (µM)

35


50

0

0

5

10

Luteolin-7-O-glucuronide concentration (µM)


Page 36 of 37

Figure 5 c

4500

IL-6

IL-1β 700

d cd

600

bc

500

3000

ab ab

1500

mRNA (fold)

mRNA (fold)

bc

400 300

ab

200 100

a

a 0

a

a

0

NF-κB1 9

Ccl2

c

c

c

40

bc

abc ab

3

mRNA (fold)

mRNA (fold)

30 6

bc 20

ab ab 10

a

a 0

a

0

Ccl3 d

30

Ccl5 30

cd

c c

c

20

b

10

mRNA (fold)

mRNA (fold)

c 20

b 10

ab

a

a

0

0

JunB 30

b

mRNA (fold)

b ab

20

ab ab

10

a 0

36


a

Page 37 of 37


TOC Graphic

37


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