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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
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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
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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
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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
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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
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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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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
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Luteolin-3’-O-glucuronide,
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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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Preparation
of
test
medium.
Test
samples
(luteolin-3’-O-glucuronide,
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
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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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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
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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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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
luteolin-4’-O-glucuronide
210
luteolin-3’-O-glucuronide and luteolin-7-O-glucuronide were extracted from rosemary
211
and
212
Luteolin-4’-O-glucuronide
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
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study,
purified.15,17
luteolin
with
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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
Luteolin-4’-O-glucuronide
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
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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
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decreased.
S1).
These
In
the
plasma
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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
luteolin-3’-O-glucuronide
16
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mechanism
(Figure
3B)
of
for
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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
luteolin-3’-O-glucuronide,
luteolin-4’-O-glucuronide,
luteolin-7-O-glucuronide
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
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or
Journal of Agricultural and Food Chemistry
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
luteolin-3’-O-glucuronide,
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
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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
luteolin-3’-O-glucuronide
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
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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
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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
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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.;
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Roussos, C.; Theodore, F. Luteolin reduces lipopolysaccharide-induced lethal toxicity
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and expression of proinflammatory molecules in mice. Am. J. Respir. Criti. Care Med.
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2002, 165, 818-823.
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(5) Androutsopoulos, V.P.; Spandidos, D.A. The flavonoids diosmetin and luteolin
355
exert
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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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Park,
C.M.;
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Luteolin
inflammatory
and
luteolin-7-O-glucoside
responses
through
26
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inhibit
modulation
of
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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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TRAIL-induced apoptosis. Life Sci. 2014, 100, 110-117.
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(33) Shimoi, K.; Nakayama, T. Glucuronidase deconjugation in inflammation. Methods
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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.
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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
luteolin-4’-O-glucuronide
luteolin-3’-O-glucuronide,
(m/z
luteolin
glucoside
(luteolin-3’-O-glucuronide,
461
[M-H]-)
(B)
luteolin-4’-O-glucuronide
28
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and
or
Page 29 of 37
Journal of Agricultural and Food Chemistry
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
luteolin-3’-O-glucuronide,
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
luteolin-3’-O-glucuronide,
luteolin-4’-O-glucuronide,
of
µM
luteolin-4’-O-glucuronide,
luteolin-7-O-glucuronide
luteolin-3’-O-glucuronide,
luteolin-3’-O-glucuronide,
29
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or
luteolin-4’-O-glucuronide,
luteolin-4’-O-glucuronide,
Journal of Agricultural and Food Chemistry
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.
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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
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Figure 1
Luteolin
Luteolin-3’-O-glucuronide
Luteolin-7-O-glucoside
Luteolin-4’-O-glucuronide
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Figure 2
A
B 100
100
285.2
285.0
OH
Glucuronate O
HO
O
m/z 285
Relative intensity (%)
Relative intensity (%)
m/z 285 Luteolin-3’-O-glucuronide 461.2 133.0
0
OH
O
Luteolin-4’-O-glucuronide
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)
Relative intensity (%)
Relative intensity (%)
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)
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Figure 3
A
100
Relative intensity (%)
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
Luteolin-3’-O-glucuronide
Relative intensity (%)
Liver
Luteolin (20 mg/kg) administration
Luteolin-4’-O-glucuronide Luteolin
Luteolin-7-O-glucuronide 0 0
10
20
30
Time (min) 100
Relative intensity (%)
Kidney
Luteolin (20 mg/kg) administration
Luteolin-3’-O-glucuronide Luteolin-4’-O-glucuronide Luteolin-7-O-glucuronide
Luteolin
0 0
10
20
30
Time (min) 100
Relative intensity (%)
Small intestine
Luteolin (20 mg/kg) administration
Luteolin-4’-O-glucuronide Luteolin-7-O-glucuronide
Luteolin
Plasma luteolin-3’O-glcuronide (µM)
8
4000
Luteolin (20 mg/kg) administration
6
3000
Luteolin-7-O-glucoside (20 mg/kg) administration
4
2000
2
1000
0
Plasma luteolin-3’O-glcuronide (µg/L)
B
Luteolin-3’-O-glucuronide
0
0 1
3
6
12 Time (h)
24
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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
Cell viability (% of control)
a
0
a
140
a
Cell viability (% of control)
Cell viability (% of control)
160
b
50
Luteolin concentration (µM)
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50
0
0
5
10
Luteolin-7-O-glucuronide concentration (µM)
Journal of Agricultural and Food Chemistry
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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
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Journal of Agricultural and Food Chemistry
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