Absorption and Metabolism of Luteolin in Rats and Humans in Relation to in Vitro Anti-inflammatory Effects

Article abstract of DOI:10.1021/acs.jafc.8b03273

Luteolin is a flavonoid present in plants in the form of aglycone or glucosides. In this study, luteolin glucosides (i.e., luteolin-7-O-β-d-glucoside, luteolin-7-O-[2-(β-d-apiosyl)-β-d-glucoside], and luteolin-7-O-[2-(β-d-apiosyl)-6-malonyl-β-d-glucoside]) prepared from green pepper leaves as well as luteolin aglycone were orally administered to rats. Regardless of the administered luteolin form, luteolin glucuronides were mainly detected from plasma and organs. Subsequently, luteolin aglycone, the most absorbed form of luteolin in rats, was orally administered to humans. As a result, luteolin-3′-O-sulfate was mainly identified from plasma, suggesting that not only luteolin form but also animal species affect the absorption and metabolism of luteolin. When LPS-treated RAW264.7 cells were treated with luteolin glucuronides and luteolin sulfate (the characteristic metabolites identified from rats and humans, respectively), the different luteolin conjugates were metabolized in different ways, suggesting that such difference in metabolism results in their difference in anti-inflammatory effects.

Full text of DOI:10.1021/acs.jafc.8b03273

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Bioactive Constituents, Metabolites, and Functions
Absorption and metabolism of luteolin in rats and
humans in relation to in vitro anti-inflammatory effects
Natsumi Hayasaka, Naoki Shimizu, Toshikazu Komoda, Satoshi Mohri,
Tojiro Tsushida, Takahiro Eitsuka, Teruo Miyazawa, and Kiyotaka Nakagawa
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03273 • Publication Date (Web): 03 Oct 2018
Downloaded from http://pubs.acs.org on October 5, 2018
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Page 1 of 38
Journal of Agricultural and Food Chemistry
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Journal of Agricultural and Food Chemistry (Research Article)
Absorption and metabolism of luteolin in rats and humans in relation
to in vitro anti-inflammatory effects
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4
Natsumi Hayasaka1, Naoki Shimizu1, Toshikazu Komoda2, Satoshi Mohri2, Tojiro
Tsushida2, Takahiro Eitsuka1, Teruo Miyazawa3,4, Kiyotaka Nakagawa1*
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1 Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science,
Tohoku University, Sendai, Miyagi 980ꢀ0845, Japan
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2 School of Food, Agricultural and Environmental Sciences, Miyagi University, Sendai,
Miyagi 982ꢀ0215, Japan
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3 Food and Biotechnology Innovation Project, New Industry Creation Hatchery Center
(NICHe), Tohoku University, Sendai, Miyagi 980ꢀ8579, Japan
4 Food and Health Science Research Unit, Graduate School of Agricultural Science,
Tohoku University, Sendai, Miyagi 980ꢀ0845, Japan
*Corresponding author: Food and Biodynamic Chemistry Laboratory, Graduate School
of Agricultural Science, Tohoku University, 468ꢀ1 Aramaki Aza Aoba, Aobaꢀku, Sendai,
980ꢀ0845, Japan. Fax: +81ꢀ22ꢀ757ꢀ4417.
Eꢀmail address: nkgw@m.tohoku.ac.jp
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ABSTRACT
Luteolin is a flavonoid present in plants in the form of aglycone or glucosides. In
this study, luteolin glucosides (i.e., luteolinꢀ7ꢀOꢀβꢀDꢀglucoside,
luteolinꢀ7ꢀOꢀ[2ꢀ(βꢀDꢀapiosyl)ꢀβꢀDꢀglucoside], and
luteolinꢀ7ꢀOꢀ[2ꢀ(βꢀDꢀapiosyl)ꢀ6ꢀmalonylꢀβꢀDꢀglucoside]) prepared from green pepper
leaves as well as luteolin aglycone were orally administered to rats. Regardless of the
administered luteolin form, luteolin glucuronides were mainly detected from plasma and
organs. Subsequently, luteolin aglycone, the most absorbed form of luteolin in rats, was
orally administered to humans. As a result, luteolinꢀ3´ꢀOꢀsulfate was mainly identified
from plasma, suggesting that not only luteolin form, but also animal species affect the
absorption and metabolism of luteolin. When LPSꢀtreated RAW264.7 cells were treated
with luteolin glucuronides and luteolin sulfate (the characteristic metabolites identified
from rats and humans, respectively), the different luteolin conjugates were metabolized
in different ways, suggesting that such difference in metabolism results in their
difference in antiꢀinflammatory effects.
Keywords
Luteolin aglycone, Luteolin glucuronide, Luteolin sulfate, Metabolism,
Antiꢀinflammatory effects
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1. INTRODUCTION
People consume substantial amounts of flavonoids through the intake of plant
foods and beverages.1ꢀ4 Intake of flavonoids from such sources is believed to be
associated with decreased risk of lifeꢀstyle related diseases such as atherosclerosis,
diabetes, and cancer.5–7 In order to unravel the mechanisms by which dietary flavonoids
exhibit their physiological effects, it is important to understand the factors that
determine their release from foods, their extent of absorption, and their metabolic fate in
vivo.8 Hence, many studies concerning the absorption and metabolism of certain major
flavonoids (e.g., catechin and quercetin) have been conducted.9,10
Luteolin is a typical flavonoid present in celery, green pepper, and perilla leaf,
found in the form of either aglycone or glucosides (e.g., luteolinꢀ7ꢀOꢀβꢀDꢀglucoside
(Lꢀ7G), luteolinꢀ7ꢀOꢀ[2ꢀ(βꢀDꢀapiosyl)ꢀβꢀDꢀglucoside] (Lꢀ7AG), and
luteolinꢀ7ꢀOꢀ[2ꢀ(βꢀDꢀapiosyl)ꢀ6ꢀmalonylꢀβꢀDꢀglucoside] (Lꢀ7AMG)) (Fig. 1A).8,11
Intake of luteolin is known to contribute to various beneficial effects such as
antiꢀinflammatory, antioxidative, and anticancer activities.2,12ꢀ14 Nevertheless, only few
studies have examined the absorption and metabolic fate of luteolin,15 which contrasts
with the case of catechin and quercetin.9,10
In one of such studies, the absorption of luteolin was examined by Lin et al.
through the oral administration of luteolin aglycone and Lꢀ7G to rats.16 Whereas
luteolin aglycone was directly absorbed, Lꢀ7G was mainly hydrolyzed to luteolin
aglycone in the gastrointestinal tract, followed by absorption into systemic circulation.
Also, Shimoi et al. reported that orally administered luteolin aglycone is mainly
metabolized to luteolin glucuronide in rats.1,8 In a similar way, when Lꢀ7G was orally
administered to rats, Lꢀ7G was first hydrolyzed to luteolin aglycone and then converted
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mainly to luteolin glucuronide. Additionally, we recently investigated the position at
which glucuronic acid conjugates with luteolin after the oral administration of luteolin
aglycone and Lꢀ7G to rats, and reported that luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide was the
major metabolite found in plasma.17 Thus, luteolin conjugates, especially
luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide, but not luteolin aglycone and luteolin glucosides, are
presumably responsible for the physiological effects of luteolin in vivo. However, the
absorption and metabolism of luteolin glucosides other than Lꢀ7G (e.g., Lꢀ7AG and
Lꢀ7AMG) is still unknown, possibly due to the difficulties in preparing such glucosides.
In addition, most studies investigating the absorption of luteolin have focused on
rodents, and human data are scarcely available.
In this study, we prepared luteolin glucosides (i.e., Lꢀ7G, Lꢀ7AG, and Lꢀ7AMG)
from green pepper leaves, and orally administered either these glucosides or luteolin
aglycone to rats, in order to investigate the differences with regard to the extent of their
absorption and metabolism. We also conducted a human test by which the most easily
absorbed form of luteolin in rats (i.e., luteolin aglycone) was orally administered to
healthy individuals, and found for the first time that not only the form of luteolin, but
also animal species affect the absorption and metabolism of luteolin. To evaluate the
relationship of this finding with the physiological effects of luteolin, luteolin
metabolites characteristic to rats and humans were each investigated for their effects on
inflammation in LPSꢀtreated RAW264.7 cells. The results of this study may help
elucidate the relationship between the biological availability and physiological effects of
luteolin in animals and humans.
2. MATERIALS AND METHODS
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2.1 Chemicals
Luteolin was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
LPS (Escherichia coli 0111) was purchased from Sigma (St. Louis, MO, USA).
Luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide, luteolinꢀ4´ꢀOꢀβꢀDꢀglucuronide, and
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luteolinꢀ7ꢀOꢀβꢀDꢀglucuronide were prepared as reported previously.18ꢀ20 For the
preparation of luteolinꢀ3´ꢀOꢀsulfate, 286 mg luteolin was dissolved in 4 mL of
anhydrous N,Nꢀdimethylformamide, of which was reacted with 318 mg of sulfur
trioxide pyridine complex for five hours at room temperature. After addition of 20 mL
water, the solution was loaded onto a TOYOPEARL HWꢀ40F column (TOSOH
Corporation, Tokyo, Japan). The column was washed with water (200 mL) and eluted
with 30% methanol (1 L). The fraction containing luteolinꢀ3´ꢀOꢀsulfate was collected,
neutralized with 1.0 M NaOH to pH 6.8, and evaporated to obtain luteolinꢀ3´ꢀOꢀsulfate
sodium salt. The purity and chemical structures of luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide,
luteolinꢀ4´ꢀOꢀβꢀDꢀglucuronide, luteolinꢀ7ꢀOꢀβꢀDꢀglucuronide, and luteolinꢀ3´ꢀOꢀsulfate
was confirmed with MS/MS analysis using a micrOTOFꢀQ II mass spectrometer
(Bruker Daltonik, Bremen, Germany) and with 1H and 13C NMR using a
JNMꢀECXꢀ400P spectrometer (Japan Electronic Co., Ltd., Tokyo, Japan) at 400ꢁMHz
with dimethyl sulfoxideꢀd6 as the solvent. Other chemicals used were of analytical grade
or higher.
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2.2 Preparation of luteolin glucosides
Green pepper leaves (500 g) were crushed with 1 L of methanol containing 0.5%
phosphoric acid. After the addition of 500 mL distilled water, the solution was filtered
under reduced pressure. A further 2 L of distilled water was added to the obtained
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filtrate. The solution was loaded onto a DIAIONTM HP20 column (Mitsubishi Chemical
Co., Ltd., Tokyo, Japan), which was then washed with 25% methanol (1 L). Lꢀ7AMG
was eluted with 2 L of 90% ethanol. The eluate was evaporated, dissolved in methanol,
and centrifuged at 1,000×g for 5 min at 4°C. The precipitate was collected and
freezeꢀdried to obtain Lꢀ7AMG powder (1.9 g).
Lꢀ7G was prepared from Lꢀ7AMG as follows: Lꢀ7AMG (0.5 g) was dissolved in
2.0 M NaOH (20 mL) and hydrolyzed at room temperature for 10 min. Subsequently,
7.5 mL of 6.0 M HCl was added, and Lꢀ7AMG was further hydrolyzed at 80°C for 1 h.
This solution was loaded onto a TOYOPEARL HWꢀ40C column (TOSOH Corporation),
and was washed with 100 mL of 40% methanol. Lꢀ7G was fractionated by gradient
elution with 70–100% methanol containing 0.1% formic acid (2 L). The fractionated
solution was dried to obtain Lꢀ7G powder (0.3 g).
For the preparation of Lꢀ7AG, Lꢀ7AMG (0.5 g) was dissolved in 1.0 M NaOH (20
mL) and hydrolyzed at room temperature for 10 min. After neutralization with 3.3 mL
of 6.0 M HCl, this solution was centrifuged at 7,700×g for 5 min at 4°C. The
precipitates were purified with distilled water and recentrifuged. The obtained
precipitate was dehydrated with methanol. After adding an excessive volume of ethyl
acetate, the solution was centrifuged twice at 7,700×g for 5 min at 4°C. Finally, the
collected precipitate was dried to obtain Lꢀ7AG powder (0.3 g).
The purity and chemical structures of Lꢀ7G, Lꢀ7AG, and Lꢀ7AMG were confirmed
by MS/MS and by 1H and 13C NMR.
2.3 Animal study
Eightꢀweekꢀold male SpragueꢀDawley rats (weight = 208 ± 12 g) were obtained
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from CLEA Japan, Inc. (Tokyo, Japan). Rats were housed in cages maintained at 23°C
with a 12 h light/dark cycle and were given free access to water and commercial rodent
chow (CEꢀ2; CLEA Japan, Inc.) for 1 week before the experiments. After 12 h fast, rats
(n = 7–8) were administered either luteolin aglycone, Lꢀ7G, Lꢀ7AG, or Lꢀ7AMG (20
mg/kg body weight) by oral gavage using 1% sodium cholate as vehicle. After 0, 1, 3, 6,
12, and 24 h of administration, blood (about 0.3 mL) was collected from the tail vein
using capillary tubes. Blood was centrifuged at 1,000×g for 15 min at 4°C to obtain
plasma. In a separate experiment, livers, kidneys, and small intestines (n = 7–8) were
excised 6 h after oral administration of either luteolin aglycone, Lꢀ7G, Lꢀ7AG, or
Lꢀ7AMG (20 mg/kg body weight). The collected samples were stored at −80°C until
analysis. The protocols of the animal study were designed in accordance with the animal
experiment guidelines and reviewed by the ethics committee of Tohoku University
(approval number: 2016ꢀnoudouꢀ006).
2.4 Human study
Nine healthy male subjects aged 23–47 years participated in the study and gave
written informed consent in accordance with the Declaration of Helsinki. The subjects
were refrained from consuming foods containing luteolin (e.g., paprika) for one week
prior to the study. Subjects orally consumed 50 mg luteolin aglycone via capsules
containing a commercial Chrysanthemum morifolium extract (Kiku flower extractꢀP;
Oryza Oil & Fat Chemical Co., Ltd., Aichi, Japan) after fasting overnight. HPLCꢀUV
analysis confirmed that the extract contained about 10% luteolin aglycone and trace
levels of apigenin. Luteolin derivatives other than luteolin aglycone were not detected.
Blood samples were collected in heparinized tubes before and 1, 3, 6, and 12 h after
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administration. Blood was centrifuged at 1,000×g for 10 min at 4°C to obtain plasma.
The obtained plasma was stored at −80°C until analysis. During the study period,
subjects were allowed to drink water ad libitum and to take light meals (Japanese
noodles free of luteolin) at 4 and 10 h after administration of luteolin capsules. The
protocols were approved by the ethics committee of Tohoku University (approval
numbers: 16ꢀAꢀ02 and 17ꢀAꢀ05).
2.5 Extraction of luteolin metabolites from plasma and organs
Luteolin metabolites were extracted from plasma and organs as described
previously.17 Acetonitrile (300 ꢂL) was added to 100 ꢂL of rat/human plasma, and
centrifuged at 1,000×g for 10 min at 4°C. After collecting the supernatant by
decantation, methanol (300 ꢂL) was added to the precipitate. The solution was mixed
and centrifuged at 1,000×g for 10 min at 4°C. The resultant supernatants were combined,
dried, and redissolved in 10% acetonitrile. For the extraction of luteolin metabolites
from organs, a 30% (w/v) homogenate solution was prepared by homogenizing each
organ (300 mg) in saline containing 1 mM ethylenediaminetetraacetic acid. Luteolin
metabolites were extracted from 500 ꢂL of the homogenate in the same way as plasma.
2.6 Cell study
Mouse macrophage RAW264.7 cells were purchased from DS Biomedical Co., Ltd.
(Osaka, Japan). RAW264.7 cells were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS, Dainippon
Sumitomo Pharmaceutical, Osaka, Japan), 1 mM sodium pyruvate, 0.1 mM nonessential
amino acids, penicillin (100 U/mL), and streptomycin (100 ꢂg/mL) at 37°C under 5%
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CO2.
RAW264.7 cells (5.0 × 105) were preincubated with 10% FBS/DMEM in 10 cm
dishes for 24 h. Luteolin aglycone and conjugates were dissolved in dimethyl sulfoxide
(50 mM), and 5 ꢂL of the solution was added to the test medium to achieve a final
concentration of 25 ꢂM. Cells were incubated in each test medium for 24 h. After
removal of cell medium, the cell surface was washed with PBS several times, after
which the cells were collected and homogenized. Intracellular luteolin metabolites were
extracted from the cell suspension in the same way as human plasma.
For the identification of genes related to the antiꢀinflammatory effects of luteolin,
RAW264.7 cells (8.0 × 105) were treated either with or without luteolin aglycone and
conjugates. After incubation for 24 h, LPS was to the medium to achieve a final
concentration of 1 ꢂg/mL. Cells were further incubated for 3 h and total RNA was
extracted with the use of an RNeasy Mini Kit (Qiagen, Tokyo, Japan). As a control,
total RNA was extracted from cells that were not treated with luteolin nor LPS.
PrimeScript Master mix (Takara Bio, Otsu, Japan) was used for the synthesis of cDNA
from total RNA (500 ng) according to the manufacturer’s instructions. PCR
amplification was performed with a CFX96 RealꢀTime PCR Detection System (BioꢀRad
Laboratories, New South Wales, Australia) with SYBR Premix Ex TaqII (Takara Bio)
and geneꢀspecific primers (SigmaꢀAldrich Japan, Tokyo, Japan) for interleukinꢀ6 (ILꢀ6),
interleukinꢀ1 beta (ILꢀ1β), CC chemokine ligand 2 (Ccl2), CC chemokine ligand 3
(Ccl3), and glyceraldehydeꢀ3ꢀphosphate dehydrogenase (GAPDH). PCR conditions
were 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 55°C for 30 s.
2.7 LC-MS/MS analysis
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LCꢀMS/MS analysis of luteolin was performed based on our previous method with
slight modifications.17 The extracted samples (10 ꢂL) were injected into a 4000 QTRAP
mass spectrometer (SCIEX, Redwood City, CA, USA) equipped with a Prominence
HPLC system (Shimadzu, Kyoto, Japan). Samples were analyzed with a C18 column
(CAPCELLPAK C18 MGII S3, 4.6×150 mm; Shiseido, Tokyo, Japan) maintained at
40°C with a flow rate of 0.8 mL/min. Gradient elution was performed using a
twoꢀsolvent system: A, water containing 0.1% trifluoroacetic acid; and B, acetonitrile.
The gradient program was as follows: 0–20 min, 90–70% A linear; 20–25 min, 70–50%
A linear. The mobile phase was split at the postcolumn, such that the eluate entered the
HPLCꢀMS/MS system at a flow rate of 0.2 mL/min. MS/MS parameters were optimized
with luteolin aglycone, luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide, luteolinꢀ4´ꢀOꢀβꢀDꢀglucuronide,
luteolinꢀ7ꢀOꢀβꢀDꢀglucuronide, and luteolinꢀ3´ꢀOꢀsulfate standards under electrospray
ionization in the negative ion mode. The MS parameters were as follows: turbo gas
temperature, 600°C; spray voltage, −4500 V; nebulizer gas, 40 psi; auxiliary gas, 40 psi;
curtain gas, 20 psi; collision gas, 4.0 psi. For the quantification of luteolin aglycone and
luteolin glucuronides, the instrument was operated in the multipleꢀreaction monitoring
(MRM) mode and concentrations were calculated based on respective standard curves.
Detection limits were in the range of 0.1−0.2 pmol/injection at a S/N ratio of 3, and
recovery tests confirmed that recoveries were >70%.17 The precursor/product ion
transitions were the following: luteolin aglycone, m/z 284 > 133 (collision energy (CE),
−48 V; declustering potential (DP), −100 V); luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide, m/z 461 >
285 (CE, −32 V; DP, −95 V); luteolinꢀ4´ꢀOꢀβꢀDꢀglucuronide, m/z 461 > 285 (CE, −32
V; DP, −95 V); luteolinꢀ7ꢀOꢀβꢀDꢀglucuronide, m/z 461 > 285 (CE, −32 V; DP, −95 V);
luteolinꢀ3´ꢀOꢀsulfate, m/z 365 > 285 (CE, −50 V; DP, −90 V). Luteolin of other forms
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were qualitatively analyzed using the following MRM transitions either defined in
literature21ꢀ23 or predicted MRM ion pairs (CE, −50 V; DP, −90 V): luteolin glucoside,
m/z 447 > 285; luteolin diglucoside, m/z 609 > 285; luteolin diglucuronide, m/z 637 >
285; luteolin glucoside glucuronide, m/z 632 > 285; luteolin disulfate, m/z 444 > 285;
luteolin glucoside sulfate, m/z 637 > 285; luteolin glucuronide sulfate, m/z 541 > 285;
methylated luteolin, m/z 301 > 285; methylated luteolin glucoside, m/z 463 > 285;
methylated luteolin glucuronide, m/z 477 > 285; methylated luteolin sulfate, m/z 381 >
285.
2.8 Statistical Analysis
Data are presented as mean ± standard error (SE). Statistical analysis was
performed by oneꢀway ANOVA followed by Tukey’s test. A p value of <0.05 was
considered to be significant.
3. RESULTS AND DISCUSSION
3.1 Preparation of luteolin glucosides for oral administration studies
Unlike catechin and quercetin, which are flavonoids that have been investigated in
detail, studies examining the metabolic fate of luteolin are limited.15 We previously
identified the structures of the major luteolin metabolites (i.e., luteolin glucuronides)
found in plasma and organs after oral administration of luteolin aglycone or Lꢀ7G to
rats,17 and suggested that luteolin conjugates, especially luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide,
are responsible for the biological activity of luteolin in vivo. However, the absorption
and metabolism of Lꢀ7AG and Lꢀ7AMG, the primary glucosides detected in certain
plants such as pepper fruits,11 are still unknown. Hence, in this study, the metabolic fate
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of luteolin glucosides (i.e., Lꢀ7G, Lꢀ7AG, and Lꢀ7AMG) and luteolin aglycone was
investigated by a single oral administration test to clarify how luteolin is absorbed and
metabolized.
Since Lꢀ7AG and Lꢀ7AMG are not commercially available, these luteolin
glucosides were prepared for oral administration studies. First, Lꢀ7AMG was extracted
from green pepper leaves and chromatographically purified with a DIAIONTM HP20
column. Lꢀ7G was fractionated with a TOYOPEARL HWꢀ40C column after
hydrolyzing Lꢀ7AMG with NaOH and HCl solutions. Lꢀ7AG was produced by
hydrolyzing Lꢀ7AMG in a NaOH solution and by drying the precipitate. Each luteolin
glucoside was pure enough for the use in oral administration tests (HPLC purity > 90%).
As a point of reference, although apiosylated flavonoids (e.g., Lꢀ7AG and
apigeninꢀ7ꢀOꢀapiosylꢀglucoside) are known to be present in nature,11,24 the
apiosylꢀmalonyl type (i.e., Lꢀ7AMG) is characteristic to luteolin. It has been suggested
that the malonyl group in Lꢀ7AMG is easily dissociated in aqueous solutions; thus, it
was thought interesting to see whether such property would affect the absorption and
metabolism of Lꢀ7AMG.
3.2 Absorption and metabolism of luteolin aglycone and glucosides in rats
In the animal study, rats were orally administered 20 mg/kg body weight of either
luteolin aglycone or luteolin glucosides (i.e., Lꢀ7G, Lꢀ7AG, and Lꢀ7AMG). This dose
was comparable to other studies observing the metabolism of flavonoids, as well as our
previous study that investigated the metabolic fate of luteolin.17 In the present study,
HPLCꢀMS/MS analysis in the MRM mode was utilized to analyze various luteolin
metabolites, since the method offers advantages such as selectivity over conventional
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techniques (e.g., HPLCꢀUV).1 As a result, regardless of the form by which luteolin was
administered, luteolin glucuronides (i.e., luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide,
luteolinꢀ4´ꢀOꢀβꢀDꢀglucuronide, and luteolinꢀ7ꢀOꢀβꢀDꢀglucuronide) were mainly
detected from plasma and organs (i.e., liver, kidney, and small intestine).
Luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide was observed as the largest peak (Fig. 1B and 2).
Luteolin diglucuronide, luteolin glucuronide sulfate, and luteolin aglycone were seen at
low peak intensities, whereas luteolin glucosides, luteolin diglucosides, luteolin
glucoside glucuronide, luteolin sulfate, luteolin disulfate, luteolin glucoside sulfate,
luteolin glucuronide sulfate, methylated luteolin, methylated luteolin glucoside,
methylated luteolin glucuronide, and methylated luteolin sulfate were either found in
only trace levels or not detected, suggesting their low concentrations in plasma and
organs. Based on the present results and earlier findings,1,8,16,17 we can suggest the
following absorption and metabolism routes of luteolin. When rats receive luteolin
aglycone, a part of luteolin is absorbed from the intestine into the systemic circulation.
In case of luteolin glucosides, regardless of sugar chain type, Lꢀ7G, Lꢀ7AG, and
Lꢀ7AMG are primarily hydrolyzed to luteolin aglycone in the gastrointestinal tract and
then absorbed into the body. Following this absorption, luteolin is mainly converted to
luteolin glucuronides, especially luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide. However, further
studies are necessary to elucidate whether such hydrolysis of different luteolin
glucosides takes place in the gastrointestinal tract.
HPLCꢀMS/MS analysis of both the plasma and organs demonstrated that the
concentration of luteolin glucuronides was larger in the luteolin aglycone administrated
group than that of the luteolin glucoside administrated groups (Table 1 and 2). In
previous studies, luteolin glucoside was suggested to be hydrolyzed to luteolin aglycone
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by the bacterial flora in the intestine, and then converted to luteolin glucuronide by
phase II enzymes in the small intestine, liver, and kidney.8,15 This suggests that luteolin
aglycone is easily absorbed compared to luteolin glucoside, because luteolin aglycone
does not need to be hydrolyzed, and coincides with the results of the current study in
which oral administration of luteolin aglycone resulted in greater amounts of luteolin
metabolites in plasma and organs compared to the administration of luteolin glucosides.
With regard to the metabolism of luteolin glucosides, the concentrations of luteolin
glucuronides were typically higher in order of the Lꢀ7G group > Lꢀ7AG group >
Lꢀ7AMG group. Hence, Lꢀ7G may be hydrolyzed and absorbed easier than Lꢀ7AG or
Lꢀ7AMG. In a previous study, Chang et al. compared the absorption of
quercetinꢀ3ꢀOꢀglucoside and quercetinꢀ3ꢀOꢀgalactoside and suggested that
quercetinꢀ3ꢀOꢀglucoside is rapidly absorbed and transformed into glucuronidated
quercetin as compared to quercetinꢀ3ꢀOꢀgalactoside.25 Such results indicate that the
absorption of flavonoid glycosides relates to the hydrolysis of the type of sugar chains
attached to its aglycone. Furthermore, the withdrawal of sugar chains attached to
flavonoids have been suggested to take place as a result of enzymatic reactions. For
example, it has been reported that the glucose bound to flavonoids is hydrolyzed by
glucosidases that exist in the lower sections of the small intestine or enteric bacteria.26
These studies suggest that glucose is easily hydrolyzed, and thus Lꢀ7G is absorbed
easier than Lꢀ7AG or Lꢀ7AMG. Also, the fact that Lꢀ7AG was absorbed easier than
Lꢀ7AMG can be considered to be due to the presence of a malonyl group in Lꢀ7AMG.
Since it is known that the malonyl group in Lꢀ7AMG is easily dissociated in aqueous
solutions, further research will be necessary to identify why the malonyl group seems to
be difficult to be dissociated during the metabolism of Lꢀ7AMG in vivo.
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The concentration of luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide in plasma reached its
maximum level at 3–6 h after oral administration, and then gradually decreased (Fig. 3).
Although the absorbance of luteolin differed according to its different forms, the major
metabolite of luteolin (i.e., luteolinꢀ3´ꢀβꢀDꢀOꢀglucuronide) remained in rat plasma even
after 24 h of administration of both luteolin aglycone and luteolin glucosides (Table 1).
Chung et al. reported that the peak concentration of catechin in rat plasma was reached
at 0.14–0.18 h after ingestion, and gradually reduced to detection limits at 12 h.9
Although the study only examined the free forms of catechin, and not its conjugates, the
fact that luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide remained to about half of the maximum level
even 12 h after administration regardless of administrating luteolin aglycone or
glucosides suggests that luteolin remains for a much longer time in rat body compared
to other flavonoids. This may be due to enterohepatic recycling, of which previous
studies have demonstrated that following initial absorption of flavonoids, their phase II
metabolites (e.g., glucuronides) are excreted to the gastrointestinal tract via bile.27,28
However, further studies are necessary to prove that such mechanism prolongs the
bioavailability of luteolin.
3.3 Absorption and metabolism of luteolin aglycone in humans
As mentioned above, it was suggested that luteolin aglycone and glucosides were
mainly metabolized to luteolin glucuronide in rats. However, because intestinal
enzymatic activities differ between humans and animals,8 we considered that luteolin is
not necessarily converted to only luteolin glucuronide in humans. While human studies
related to the metabolism of other flavonoids (e.g., catechin and quercetin) have been
conducted,29ꢀ31 knowledge regarding the metabolism of luteolin in humans have been
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scarce. Therefore, we conducted a human study in which luteolin aglycone, the most
absorbed form of luteolin in the animal study, was orally administered. Nine male
subjects consumed 50 mg luteolin aglycone, and blood samples were collected over
time to investigate timeꢀdependent changes of luteolin in human plasma. As a result,
consistent with the oral administration test to rats, luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide and
luteolinꢀ4´ꢀOꢀβꢀDꢀglucuronide were detected in human plasma (Fig. 4A). However, the
maximum peak identified during the LCꢀMS/MS analysis of human plasma was not
luteolin glucuronide but was an unidentified peak. Considering the study by Roubalová
et al. in which the major form of quercetin identified in humans was
quercetinꢀ3´ꢀOꢀsulfate,32 and the fact that phenol sulfotransferase, an enzyme that
metabolizes flavonoids to flavonoid sulfates, is present in the human intestine,33 it was
speculated that the unidentified peak was luteolin sulfate. Hence, a standard of luteolin
sulfates (i.e., luteolinꢀ3´ꢀOꢀsulfate and luteolinꢀ4´ꢀOꢀsulfate) was prepared by reacting
luteolin aglycone with sulfur trioxide pyridine complex. Luteolin sulfate in human
plasma was analyzed with the use of these standards, and consequently, the unidentified
peak corresponded to that of the luteolinꢀ3´ꢀOꢀsulfate standard. Based on these results,
it was suggested that the metabolism of luteolin varies according to animal species.
Subsequently, timeꢀdependent changes of luteolinꢀ3´ꢀOꢀsulfate concentration in human
plasma were further investigated. The plasma concentration of luteolinꢀ3´ꢀOꢀsulfate
reached its maximum level (801.9 ± 277.6 nM) at 3 h and was about half of the
maximum concentration at 6 h after oral administration of luteolin aglycone. Although
the concentration gradually decreased, luteolinꢀ3´ꢀOꢀsufate still remained even after 12
h of administration (Fig. 4B). In the study by Mullen et al. where humans consumed
261 ꢂmol quercetin (equivalent to 79 mg quercetin aglycone), the concentration of
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quercetinꢀ3´ꢀOꢀsulfate in human plasma reached a maximum level (665.0 ± 82.0 nM) at
0.75 h and was half at 1.71 h after administration.31 Thus, our results suggest that
luteolin remains for a relatively long time in the human body compared with other
flavonoids. However, compared with the results of the animal study where luteolin
glucuronides remained in plasma to about half of the maximum level 12 h after
administration (Fig. 3), luteolin sulfate remained in human plasma for a considerably
shorter time (Fig. 4B). This may be related to the enterohepatic recycling discussed
above, considering that while flavonoid sulfates are mainly excreted only into urine,
glucuronides are usually excreted also to bile to undergo enterohepatic recycling.34ꢀ36
Thus, further studies examining luteolin glucuronides and sulfates in urine may provide
a more detailed view on the possible mechanism regarding how enterohepatic
circulation possibly prolongs the bioavailability of luteolin.
3.4 In vitro anti-inflammatory effects of luteolin conjugates in relation to their
cellular intake
Based on the above studies, it was elucidated that the metabolism of luteolin is
different between rats and humans. As such, a cell study was conducted to examine
whether different luteolin metabolites demonstrate different strengths of physiological
effects. Recent studies have examined the antiꢀinflammatory effects of luteolin; Chen et
al. reported that luteolin aglycone inhibits the expression of inflammatory genes (i.e.
TNFꢀα, ILꢀ6, iNOS, and COXꢀ2) in LPSꢀtreated mouse macrophage RAW264.7 cells.37
In addition, we previously compared the antiꢀinflammatory properties of luteolin
aglycone and glucuronides (i.e., luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide,
luteolinꢀ4´ꢀOꢀβꢀDꢀglucuronide, and luteoinꢀ7ꢀOꢀβꢀDꢀglucuronide) in LPSꢀtreated
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RAW264.7 cells.17 However, the antiꢀinflammatory properties of luteolin sulfate, which
was identified as the major metabolite of luteolin in humans in the current study, has not
yet been evaluated. Moreover, studies on how luteolin aglycone and luteolin conjugates
are incorporated into cells have been scarce, although it can be assumed that luteolin
first has to be incorporated into the cell to exhibit physiological functions (e.g.,
antiꢀinflammatory effects). Therefore, the antiꢀinflammatory effects of luteolin aglycone,
luteolin glucuronides (i.e., luteolin metabolites characteristic to rats), and luteolin
sulfates (i.e., luteolin metabolites characteristic to humans) were investigated in relation
to their cellular intake.
Our previous study along with other reports have demonstrated that luteolin
aglycone and luteolin conjugates at a concentration of 25 ꢂM have no effect on cell
viability.17,37 As such, we decided that a concentration of 25 ꢂM was suitable for
examining the cellular intake and antiꢀinflammatory effects of luteolin. The cellular
intake of luteolin was investigated by extracting luteolin from RAW264.7 cells treated
with either luteolin aglycone, luteolin glucuronides (i.e., luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide
and luteolinꢀ4´ꢀOꢀβꢀDꢀglucuronide), or luteolin sulfate (i.e., luteolinꢀ3´ꢀOꢀsulfate). Also,
to investigate the antiꢀinflammatory effects of each luteolin form, RAW264.7 cells were
first treated with each luteolin form for 24 h, after which LPS (1 ꢂg/mL) was added to
the test medium. After further incubating for 3 h, total RNA was extracted, and mRNA
expression of ILꢀ6, ILꢀ1β, Ccl2, and Ccl3 was examined by realꢀtime RTꢀPCR. These
genes related to inflammation were selected based on the results of our previous study,
in which it was identified through DNA microarray analysis that luteolin treatment
affects their expression.17
When RAW264.7 cells were treated with luteolin aglycone, luteolin aglycone was
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mainly detected from the cell (Fig. 5). Also, in accordance with our previous study,17
PCR analysis demonstrated that treatment of cells with luteolin aglycone inhibits the
expression of ILꢀ6, ILꢀ1β, Ccl2, and Ccl3 genes (Fig. 6). With regard to treatment of
cells with luteolin conjugates, when cells were treated with luteolin glucuronide (i.e.,
luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide and luteolinꢀ4´ꢀOꢀβꢀDꢀglucuronide), luteolin aglycone
was the main metabolite detected from the cell. Shimoi et al. previously reported that
βꢀglucuronidase enzymes convert luteolin glucuronides to luteolin aglycone as a
consequence of inflammation in vitro in human neutrophils as well as in vivo in rats.38
The predominance of luteolin aglycone after treatment of luteolin glucuronides may be
a result of such βꢀglucuronidase activity; previous studies identifying βꢀglucuronidase
as the major deglucuronidation enzyme that deconjugates quercetin glucuronide in
RAW264 cells, further emphasizes this hypothesis.39,40 The expression levels of
inflammatory genes were also suppressed, suggesting that luteolin aglycone is the major
form related to the antiꢀinflammatory effects of luteolin. On the other hand, the
intracellular amounts of luteolin metabolites after the treatment of cells with
luteolinꢀ3´ꢀOꢀsulfate was very low compared with that of treatment with luteolin
glucuronides, suggesting that luteolinꢀ3´ꢀOꢀsulfate is difficult to be incorporated into
RAW264.7 cells. Also, metabolites other than luteolinꢀ3´ꢀOꢀsulfate were not detected
(Fig. 5), which demonstrates the difference in metabolism between luteolinꢀ3´ꢀOꢀsulfate
and luteolin glucuronides. This difference in metabolism was somewhat reflected in the
expression levels of inflammatory genes, considering that the inhibitory effects of
luteolinꢀ3´ꢀOꢀsulfate was lower than that of luteolin aglycone and luteolin glucuronide
(Fig. 6). One exception was the expression level of Ccl2, which was increased by
treatment with luteolinꢀ3´ꢀOꢀsulfate; the reason for this is unclear and requires further
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research. With regard to the physiological effects of phenolic sulfates, Roubalová et al.
reported that the bioactivity of quercetin sulfate in vitro is weak because the affinity of
sulfated quercetin to target molecules is low compared to quercetin aglycone.32
Similarly, it can be considered that the antiꢀinflammatory effects of luteolin sulfate was
weaker than that of luteolin aglycone and luteolin glucuronide due to the affinity of
luteolin sulfate to molecules that control the expression of inflammatory genes being
lower.
In conclusion, it was revealed that regardless of administrating luteolin aglycone or
glucosides, these molecules are mainly metabolized to luteolin glucuronide (e.g.,
luteolinꢀ3´ꢀOꢀβꢀDꢀglucuronide) in rats. Meanwhile, luteolinꢀ3´ꢀOꢀsulfate was identified
as the most abundant luteolin form in human plasma after administration of luteolin
aglycone. Although the metabolism of luteolin varied between animal species, it was
suggested that luteolin glucuronide and luteolin sulfate, which are the major metabolites
of luteolin in rats and humans, remain for a relativity long time in the body. Luteolin
glucuronide was mainly converted to luteolin aglycone after being incorporated into the
cell and inhibited the expression of inflammatory genes in LPSꢀtreated RAW264.7 cells.
Luteolin sulfate also reduced expression levels of inflammatory genes, but this effect
was weaker than that of luteolin glucuronide. However, further investigations will be
required to fully elucidate the mechanism by which luteolin exhibits its physiological
functions in vitro and in vivo.
Abbreviations Used
CE, collision energy; DP, declustering potential; FBS, fetal bovine serum; Lꢀ7AG,
luteolinꢀ7ꢀOꢀ[2ꢀ(βꢀDꢀapiosyl)ꢀβꢀDꢀglucoside]; Lꢀ7AMG,
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luteolinꢀ7ꢀOꢀ[2ꢀ(βꢀDꢀapiosyl)ꢀ6ꢀmalonylꢀβꢀDꢀglucoside]; Lꢀ7G,
luteolinꢀ7ꢀOꢀβꢀDꢀglucoside; MRM, multipleꢀreaction monitoring
Acknowledgements
The authors thank Shunji Kato (Division of Host Defense Mechanism, Tokai University
School of Medicine, Japan) for support in LCꢀMS/MS analysis.
Funding sources
This study was supported in part by a grantꢀinꢀaid “A scheme to revitalize agriculture
and fisheries in disaster area through deploying highly advanced technology” from the
Ministry of Agriculture, Forestry and Fisheries (MAFF), Japan.
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Tables
Table 1. Timeꢀdependent changes of luteolinꢀ3’ꢀOꢀβꢀDꢀglucuronide in rat plasma
following oral administration of luteolin aglycone or glucosides.
Luteolin aglycone administration
Lꢀ7G administration
Lꢀ7AG administration
Lꢀ7AMG administration
Time (h)
Concentrations
(nM)
Concentrations
(ꢂg/L)
Concentrations
Concentrations
(ꢂg/L)
Concentrations
Concentrations
(ꢂg/L)
Concentrations
Concentrations
(ꢂg/L)
(nM)
(nM)
n.d.*
(nM)
n.d.*
0
1
n.d.*
n.d.*
n.d.*
n.d.*
n.d.*
n.d.*
356.5 ± 499.0
1074.1 ± 909.0
570.0 ± 519.3
531.7 ± 742.9
6.8 ± 6.3
164.7 ± 230.5
496.3 ± 420.0
263.1 ± 239.9
245.6 ± 343.2
3.1 ± 2.9
94.3 ± 91.0
301.1 ± 153.8
153.8 ± 156.4
133.2 ± 185.0
4.3 ± 5.3
43.6 ± 42.0
139.1 ± 195.5
71.0 ± 72.3
61.5 ± 85.5
2.0 ± 2.4
25.3 ± 16.6
24.3 ± 14.8
121.2 ± 147.4
11.0 ± 8.6
1.3 ± 0.8
11.7 ± 7.7
11.2 ± 6.8
56.0 ± 68.1
5.1 ± 4.0
0.6 ± 0.4
1.2 ± 0.5
2.8 ± 1.5
40.5 ± 72.9
30.8 ± 27.7
0.5 ± 0.5
0.6 ± 0.2
1.3 ± 0.7
18.7 ± 33.7
14.2 ± 12.8
0.2 ± 0.2
3
6
12
24
Data are mean ± SE (n = 7ꢀ8).
Lꢀ7G, Luteolinꢀ7ꢀO ꢀglucoside; Lꢀ7AG, Luteolinꢀ7ꢀO ꢀapiosylꢀglucoside; Lꢀ7AMG, Luteolinꢀ7ꢀO ꢀapiosylꢀmalonylꢀglucoside.
*n.d. = not detected
615
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618
Table 2. The concentration of luteolin glucuronides in rat organs at 6 h after oral
administration of luteolin aglycone or glucosides.
Luteolinꢀ3'ꢀO ꢀglucuronide
Luteolinꢀ4'ꢀO ꢀglucuronide
Luteolinꢀ7ꢀO ꢀglucuronide
Concentration
(pmol/g)
Concentration
(ng/g)
Concentration
(pmol/g)
Concentration
(ng/g)
Concentration
(pmol/g)
Concentration
(ng/g)
Liver
638.2 ± 344.8
906.7 ± 344.3
294.8 ± 159.3
418.9 ± 159.1
277.5 ± 173.1
184.6 ± 87.9
597.5 ± 440.9
150.4 ± 109.7
50.8 ± 38.3
92.6 ± 98.8
16.5 ± 12.7
12.1 ± 11.0
50.5 ± 84.5
10.0 ± 6.0
128.2 ± 80.0
85.3 ± 40.6
276.0 ± 203.7
69.5 ± 50.7
23.5 ± 17.7
42.8 ± 45.6
7.6 ± 5.9
165.1 ± 163.5
120.2 ± 86.8
76.3 ± 65.4
55.6 ± 34.7
Luteolin aglycone
administration
Kidney
Small
intestine
4818.5 ± 7008.5 4542.8 ± 3237.9
3573.4 ± 2814.4 1650.9 ± 1126.0
Liver
186.7 ± 91.0
258.7 ± 220.9
487.8 ± 443.0
41.7 ± 58.6
86.2 ± 42.1
119.5 ± 102.1
225.4 ± 204.7
19.3 ± 27.1
22.6 ± 17.6
97.6 ± 151.4
12.2 ± 10.1
15.0 ± 12.0
142.4 ± 198.8
23.1 ± 8.8
32.6 ± 26.2
339.5 ± 375.7
13.6 ± 10.1
13.1 ± 10.9
149.2 ± 246.1
6.0 ± 5.1
10.7 ± 4.1
15.1 ± 12.1
142.8 ± 185.4
6.3 ± 4.7
Lꢀ7G
administration
Kidney
Small
intestine
Liver
Lꢀ7AG
administration
Kidney
49.0 ± 38.2
5.6 ± 5.1
6.0 ± 5.0
Small
intestine
211.2 ± 327.6
26.5 ± 25.1
23.4 ± 39.1
4.6 ± 2.8
68.9 ± 113.7
2.8 ± 2.4
Liver
Lꢀ7AMG
administration
Kidney
33.5 ± 30.0
6.8 ± 5.8
3.1 ± 2.7
6.9 ± 8.0
3.2 ± 3.7
Small
intestine
308.2 ± 496.8
39.5 ± 54.2
18.2 ± 25.0
312.4 ± 517.2
144.3 ± 238.9
Data are mean ± SE. (n = 4)
Lꢀ7G, Luteolinꢀ7ꢀO ꢀglucoside; Lꢀ7AG, Luteolinꢀ7ꢀO ꢀapiosylꢀglucoside; Lꢀ7AMG, Luteolinꢀ7ꢀO ꢀapiosylꢀmalonylꢀglucoside.
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