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The Bioactive Flavonoid Taxifolin Inhibits Differentiation and
the Production of the Inflammatory Cytokine Interleukin-6 in
Cultured Human Adipocytes
Sakurai M1*, Hu A2, Yamaguchi T2, Tabuchi M2, Ikarashi Y2, Naraoka Y3
and Kobayashi H1,2
Department of Hospital Administration, Juntendo University Graduate School of Medicine,Japan
2
Department of Personalized Kampo Medicine, Juntendo University Graduate School of
Medicine, Japan
3
Intractable Disease Research Center, Juntendo University School of Medicine, Japan
1

Research Article
Volume 7 Issue 1
Received Date: February 15, 2022
Published Date: March 03, 2022
DOI: 10.23880/apct-16000195*Corresponding author: Mami Sakurai, Department of Hospital Administration, Juntendo University Graduate School of
Medicine, 2-1-1-Hongo, Bunkyo-ku, Tokyo 113-8421, Japan, Tel: +81-3-3813-3111 (ext. 2115); Fax: +81-3-3814-8040; Email:ma-sakurai@juntendo.ac.jp

Abstract
Background: Taxifolin, a bioactive flavonoid that possesses potent antioxidant activity, has been reported to show
multiple pharmacological properties, including protective effects against obesity-related diabetic nephropathy and diabetic
cardiomyopathy. However, knowledge regarding the effects of taxifolin on adipocytes, which are closely associated withobesity and diabetes, is insufficient.
Objective: This study aimed to explore the direct effects of taxifolin on differentiation and inflammation adipocytes byculturing human preadepocytes (HPAds).
Methods: HPAds were cultured for 16 days in a differentiation medium with or without taxifolin to examine its effect on
differentiation. On day 16, levels of lipid and differentiation-related gene expression (PPARγ, C/EBPα, adiponectin, CD36, and
GLUT4 mRNAs) in adipocytes were measured using the Oil Red O assay and the real-time polymerase chain reaction assay,
respectively. Adiponectin levels in the medium were measured using an enzyme-linked immunosorbent assay (ELISA). The
taxifolin effect on inflammation was assessed using mature adipocytes differentiated for 15 days. After incubating matureadipocytes in a differentiation medium containing tumor necrosis factor-α (TNF-α) or TNF-α + taxifolin for three hours, thelevel of interleukin-6 (IL-6), an inflammatory cytokine in the medium, was measured using ELISA.
Results: Exposure of taxifolin to adipocytes during differentiation decreased the levels of lipid in adipocytes and adiponectinin the medium. It also decreased the expression levels of C/EBPα, adiponectin, CD36, and GLUT4 mRNAs, but not PPARγmRNA. Taxifolin inhibited the increase in IL-6 levels in the medium induced by TNF-α in mature adipocytes.
Conclusion: These results suggest that taxifolin has anti-differentiation and anti-inflammatory effects on adipocytes.
Additionally, taxifolin is expected to have the potential as a therapeutic drug for obesity and metabolic syndrome.

Keywords: Taxifolin; Adipocyte; Differentiation; Inflammation; Adiponectin; IL-6
The Bioactive Flavonoid Taxifolin Inhibits Differentiation and the Production of the Inflammatory
Cytokine Interleukin-6 in Cultured Human Adipocytes

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Advances in Pharmacology and Clinical Trials

Abbreviations: MCP-1: Monocyte Chemoattractant

Protein-1; IL-6: Interleukin-6; TNF-α: Tumor Necrosis
Factor-α; NFκB: Nuclear Factor-κB; PPARγ: Peroxisome
Proliferator-Activated Receptor γ; PDE: Phosphodiesterase;
LPS: Lipopolysaccharide; DMSO: Dimethyl Sulfoxide; HPAds:
Primary Human Preadipocytes; CCK-8: Cell Counting Kit-8;
C/EBP: CCAAT/Enhancer-Binding Protein; GLUT4: Glucose
Transporter Type 4; PCR: Polymerase Chain Reaction;
ANOVA: Analysis of Variance.

Introduction

Obesity, especially visceral fat obesity, is commonly
associated with type 2 diabetes, coronary artery disease,and hypertension, and the coexistence of these diseases iscalled metabolic syndrome [1,2]. Obesity is a pathologicallyexcessive accumulation of body fat, and its prevalence
has recently increased dramatically [3-5]. Particularly, theaccumulation of visceral fat induces metabolic disorders,such as glycolipids, increasing the risk of developing insulin
resistance and diabetes. Additionally, diabetes increases
the risk of developing complications, such as diabetic
retinopathy, nephropathy, and neuropathy. It also increases
the risk of developing life-threatening arteriosclerosis, suchas myocardial and cerebral infarctions [6-9].

Adipose tissue is a storage organ that stores excess energy
and an endocrine organ that produces and secretes various
adipocytokines. Normal and healthy adipocytes promote
glycolipid metabolism [10,11]. However, obese adipose
tissue is excessively enlarged by a combination of increased
size (hypertrophy) and number (hyperplasia) of adipocytes[5]. In addition to excessive lipid accumulation, hypertrophic
adipocytes produce and secrete proinflammatory cytokinesand chemokines, such as interleukin-6 (IL-6) and monocyte
chemoattractant protein-1 (MCP-1) [12]. During thedevelopment of obesity, macrophage infiltration into adipose
tissue increases, and macrophage-derived tumor necrosis
factor-α (TNF-α) enhances proinflammatory cytokines
production in adipocytes [13-15]. That is, the macrophagederived TNF-α activates the nuclear factor-κB (NFκB) in
hypertrophic adipocytes and induces the synthesis andsecretion of proinflammatory cytokines such as IL-6 and
MCP-1 [13,14,16] However, the production and secretionof the anti-inflammatory adipocytokine adiponectin, which
inhibits the NFκB pathway, is conversely reduced in obese
hypertrophic adipocytes [15,17]. Such regulatory disruptionof adipocytokine production in hypertrophic adipocytes isthought to cause chronic inflammatory conditions, leading
to insulin resistance and diabetes [6]. Therefore, reducing
hypertrophic adipocytes and promoting the normal functionsof adipocytes are crucial for treating and preventing various
diseases caused by obesity and metabolic syndrome.

Many pharmacological approaches have been reported
to treat obesity-related diabetes and insulin resistance.
For example, peroxisome proliferator-activated receptor γ(PPARγ) agonists, such as thiazolidine derivatives promoteadipocyte differentiation [18-20], and increase adiponectin
production, exerting an anti-inflammatory effect bysuppressing the TNF-α-activated NFκB pathway [13,14,16].
AdipoRon, an adiponectin receptor agonist, has beenreported to bind to the adiponectin receptors AdipoR1 andAdipoR2, and exert antidiabetic effects through activationof AMP-activated protein kinase and PPAR-α pathways,
respectively [21,22]. Conversely, xanthine derivatives,
such as theobromine and theophylline, suppress adipocyte
differentiation and promote lipolysis by inhibiting adenosine
receptor and phosphodiesterase (PDE), causing an antiobesity effect [23,24].

Taxifolin (dihydroquercetin), is a bioactive flavonoid
found in grapes, citrus fruits, onions, green tea, olive oil,and several herbs, such as milk thistle, French maritime
bark, Douglas fir bark, and Smilacis Glabrae Rhizoma[25]. Taxifolin has a strong antioxidant activity and has
shown promising pharmacological properties in managing
inflammation, tumors, microbial infections, oxidative stress,
dementia, cardiovascular, and liver disorders [26]. Recently,
in addition to the multiple pharmacological properties,
taxifolin was also reported to prevent diabetic nephropathy
in rats [27] and diabetic cardiomyopathy in mice [28].
These reports suggest that taxifolin may be effective indiabetes management. However, there are few studies
on adipocytes that are closely related to diabetes, but theeffects of taxifolin on inflammatory and lipid metabolism incultured adipocytes and macrophages were reported more
recently [29]. The results showed that 1) taxifolin inhibited
lipopolysaccharide (LPS)-induced nitric oxide production
and tert-butyl hydroperoxide-induced reactive oxygen
species production as well as LPS-induced expressionof IL-1β and TNF-α mRNAs, in a mouse macrophage-likecell line, RAW264.7 cells. 2) Taxifolin increased the liver
X receptor β mRNA expression, involved in preventing
hyperlipidemia in macrophage-differentiated THP-1 cells.
Additionally, 3) taxifolin reduced lipid accumulation in
adipocyte-differentiated 3T3-L1 cells. These results suggestthat taxifolin may improve obesity-related oxidative
stress, inflammation, and lipid metabolism by acting on
macrophages in obese adipose tissue. However, knowledge
regarding the direct effects of taxifolin on adipocytes isinsufficient. That is, there is only evidence that taxifolin
reduced lipid accumulation in 3T3-L1 cells.
Therefore, this study aimed to clarify the direct effect of
taxifolin on adipocytes. To clarify the objectives, the effectsof taxifolin on adipocyte differentiation and TNF-α-induced

Sakurai M, et al. The Bioactive Flavonoid Taxifolin Inhibits Differentiation and the Production of theInflammatory Cytokine Interleukin-6 in Cultured Human Adipocytes. Adv in Phar & Clin Tria 2022,
7(1): 000195.

Copyright© Sakurai M, et al.

3Advances in Pharmacology and Clinical Trials

inflammatory cytokine IL-6 using cultured human adipocytes
were examined.

Materials and Methods

Test Substances and Agents
Taxifolin (Da Hurian Larch Extract Dihydroquercetin,
98.7% purity, Ametis JSC, Amur Region, Russia) was supplied
from DHQ Co., Ltd. (Saitama, Japan). TNF-α and dimethyl
sulfoxide (DMSO) were purchased from Fujifilm Wako Pure
Chemical Corporation (Osaka, Japan) and Sigma-Aldrich (St.
Louis, MO, USA), respectively. Oil Red O and 2-propanol werepurchased from Wako (Osaka, Japan) and Sigma-Aldrich(St. Louis, MO, USA). Other chemicals were purchased fromcommercial sources.

Cell Culture for Preadipocyte Proliferation

Primary human preadipocytes (HPAds) derived fromhuman pericardiac adipose tissue (Cell Applications, Inc.,San Diego, CA, USA) were seeded into a 75 cm2 culture flask(Corning, Corning, NY, USA) with the preadipocyte growth
medium, including 10% FBS, 10 μg/mL insulin, 100 U/
mL penicillin, 100 μg/ml streptomycin, and 250-ng/ml
amphotericin B (Cat No. 811K-500, Cell Applications, Inc),and cultured at 37°C in an atmosphere with 5% CO2 until
it attained confluence. During growth culture, the growth
medium was changed every two days.

Experimental Designs

Cell Viability: The confluent HPAds(16,500 cells/well)
were seeded into a collagen-coated 96-well microplate(AGC TECHNO GLASS Co., Ltd., Shizuoka, Japan) containing
preadipocyte growth medium (150 μL/well), and cultured
at 37°C and 5% CO2 until 100% confluence. Confluent cells
were used to determine adipocytes cell viability exposed totaxifolin for 16 d and three h.

Cytotoxicity for 16 days during differentiation:
Differentiation of confluent cells cultured in 96-well
microplates were initiated (Day 0) by replacing the growth
medium with the adipocyte differentiation medium,including 10% FBS, 5 μg/mL insulin, 100 U/mL penicillin,
100 μg/ml streptomycin, and 250 ng/ml amphotericin B,
0.5 μM dexamethasone, 0.25 mM IBMX (Cat No. 811D-250,
Cell Applications, Inc.) or the medium containing various
taxifolin concentrations (final concentrations: 0.2, 2, and10 µg/mL taxifolin dissolved 0.5% DMSO). The cells were
cultured at 37°C with 5% CO2 during differentiation. The
differentiation medium was changed every three days. On
day 16 of differentiation, cell viability was measured using acell counting kit-8 (CCK-8) assay.

Three hours cytotoxicity against differentiated matureadipocytes on day 15: Confluent HPAds prepared following
the procedure described in the previous section were
cultured for 15 d in the differentiation medium. On day 15
of differentiation, the culture medium was replaced with afresh differentiation medium including 0.5% DMSO (control)or the medium containing various concentrations of taxifolin(final concentrations: 0.2, 2, and 20 µg/mL) and TNF-α (5ng/mL) dissolved in 0.5% DMSO, and then the cells wereincubated at 37°C with 5% CO2. After 3h-incubation, cell
viability was measured using a CCK-8 assay.

CCK-8 assay: For cell viability determination, 15 μL of
the CCK-8 solution (Dojindo Molecular Technologies,
Gaithersburg, MD, USA) was added to wells containing cells
exposed to taxifolin for 16 days and three hours. The cells
were incubated for another 1–2 h at 37°C in a CO2 incubatorand measured at 450 nm using a microplate reader (Thermo
Fisher Scientific, Inc., Waltham, MA, USA). Cell viability wasevaluated as a percentage of the absorbance of drug-treatedcells relative to that of the control (100% viability).

Changes in Adipocyte Lipid and Medium Adiponectin
Levels during Differentiation
The confluent HPAds (42,000 cells/well) were seeded intoa collagen-coated 48-well microplate (AGC TECHNO GLASSCo., Ltd.) containing the growth medium (150 μL/well), andcultured at 37°C with 5% CO2 until 100% confluence.

Differentiation was initiated (Day 0) by replacing
the medium of the confluent preadipocytes with thedifferentiation medium (Cell Applications, Inc.). The cells
were cultured at 37°C with 5% CO2 during differentiation,and the differentiation medium was changed every threedays. The state of adipocyte differentiation was confirmed bystaining adipocytes on days zero, two, seven, 16, 31, and 37
with Oil Red O after removing the medium, and measuring
the lipid levels of adipocytes in the wells after microscopic
examination. The media obtained on days zero, two, seven,16, 31, and 37 of differentiation were used for the adiponectin
assay.

Effects of Taxifolin on Adipocyte Differentiation
Confluent HPAds prepared according to the proceduredescribed in the previous section were cultured for
16 days in the differentiation medium (control) or the
medium containing various taxifolin concentrations (final
concentrations: 0.2, 2, and 10 µg/mL) dissolved in 0.5%
DMSO. The lipid accumulation in adipocytes and theadiponectin concentration in the medium were measured on
day 16 of differentiation using Oil Red O staining and ELISA
assays, respectively, to evaluate the effect of taxifolin on
adipocyte differentiation.
To further investigate the taxifolin effect on adipocyte

Sakurai M, et al. The Bioactive Flavonoid Taxifolin Inhibits Differentiation and the Production of theInflammatory Cytokine Interleukin-6 in Cultured Human Adipocytes. Adv in Phar & Clin Tria 2022,
7(1): 000195.

Copyright© Sakurai M, et al.

4Advances in Pharmacology and Clinical Trials

differentiation, expression of differentiation-related genes[PPARγ, CCAAT/enhancer-binding protein (C/EBPα),adiponectin, cluster of differentiation 36 (CD36), and glucose
transporter type 4 (GLUT4) mRNAs] was measured on day
16 in an experiment set separately by the same procedureusing a collagen-coated 6-well microplate (AGC TECHNOGLASS Co., Ltd.) seeded with HPAds (440,000 cells/well).
Each gene expression was measured using the real-timepolymerase chain reaction (PCR) analysis.

Effects of taxifolin on TNF-α-induced proinflammatory
cytokine IL-6
The taxifolin effect on TNF-α-induced increase in IL-6 was
examined in mature adipocytes on day 15 of differentiation.
Briefly, on day 15 of differentiation, the differentiation
medium was replaced with a fresh differentiation medium
containing TNF-α (final concentration: 5 ng/mL) or TNF-αplus various taxifolin concentrations (final concentrations:0.2, 2, and 20 µg/mL) dissolved in 0.5% DMSO, and then thecells were incubated for three hours at 37°C with 5% CO2.
Controls were similarly cultured in the normal differentiation
medium. The aliquot of the culture medium was carefullycollected after incubation. The medium IL-6 concentration
was measured using ELISA kit.

Measurement of Adipocyte Lipid Stained with
Oil Red O

The adipocytes cultured in a well were rinsed twice with
500 μL phosphate-buffered saline after the medium wasremoved, then 200 μL 0.36% Oil Red O prepared with a 60%
2-propanol was added to stain intracellular lipid for 15 min atroom temperature. Then, the Oil Red O solution was removed
and the cells were washed thrice with distilled water, after
which 250 μL 2-propanol was added to extract the dye byshaking for 30 min. The extracted dye was measured using a
microplate reader (Thermo Scientific Multiskan FC, Thermo
Fisher Scientific, Inc.) at a 540 nm wavelength.

ELISA Assays for Adiponectin and IL-6

Concentrations of adiponectin and IL-6 in the medium
were measured using the AssayMaxTM human adiponectin
ELISA kit (Assaypro, LLC, St. Louis, MO, USA) andinterleukin-6 ELISA kit (Chondrex, Inc, WA, USA), following
the manufacturer’s instructions. The ELISA sensitivity was
0.391 ng/mL for adiponectin and 9 pg/mL for IL-6.

Real-Time PCR Analysis to Determine theExpression of Differentiation-Related Genes

Total RNA in adipocytes was isolated using the
FastGeneTM RNA Basic Kit (Nippon Genetics Co., Ltd.,Tokyo, Japan) following the manufacturer’s instructions. The
total RNA (2.0 μg) was reverse transcribed to cDNA using
the ReverTra AceR qPCR RT Kit (Toyobo Co., Ltd., Osaka,
Japan) following the manufacturer’s instructions. Each gene
expression was quantified with the Applied BiosystemsR
7500 fast real-time PCR system (Thermo Fisher Scientific
K.K., MA, USA) using specific primers and KOD SYBRR qPCR
Mix (Toyobo Co., Ltd.). The PCR reaction conditions were as
follows. Initial denaturation: 2 min at 98°C, the denaturation:
10 sec at 98°C, annealing: 10 sec at 60°C, extension: 30 sec
at 68°C, and the number of cycles; 40. The base sequences ofthe forward (F) and reverse (R) primers of each gene were as
follows: GAPDH: (F) 5’-CTTTGTCAAGCTCATTTCCTGGTAT-3’
and(R)
5’-TGGTGGTCCAGGGGTCTTA-3’,
PPARγ:(F)
5’-GAGCCCAAGTTTGAGTTTGC-3’
and(R)
5’-CAGGGCTTGTAGCAGGTTGT-3’,
C/EBPα:(F)
5’-GGACCCTCAGCCTTGTTTGT-3’
and(R)
5’-TGGTGGTTTAGCAGAGACGC-3’,
adiponectin:(F)
5’-AAGGAGATCCAGGTCTTATTGG-3’
and(R)
5’-ACCTTCAGCCCCGGGTAC-3’,
CD36:
(F)
5’-TCTTTGGCTTAATGAGACTGGG-3’
and(R)
5’-AAGCAACAAACATCACCACACC-3’,
and
GLUT4:(F)
5’-CGACCAGCATCTTCGAGACA-3’,
and(R)
5’-CACCAACAACACCGAGACCA-3’.
The gene expression level was expressed relative to thecontrol (=1.0) after normalization using the GAPDH gene
expression level.

Statistical Analysis

All values were represented as the mean ± S.E.M.
Dunnett’s test evaluated the statistical significance after
a one-way analysis of variance (ANOVA). The acceptedsignificance level was P < 0.05.

Results

Cell Viability
The non-cytotoxic taxifolin concentration wasdetermined by exposing taxifolin (0.2, 2, 10, and 20 µg/mL) to cells for 16 d from the onset of differentiation in adifferentiation experiment. In anti-inflammatory experiment,
taxifolin was exposed to the 15th day-mature cells for 3 h.
Cell viability is calculated as the percentage of viable cells in
the taxifolin-treated group relative to the control group, andthe range of non-toxic concentrations in each experiment isshown in Table 1. The viability of cells exposed to 0.2–10 µg/
mL taxifolin for 16 d was 83.7 ± 3.5%, The viability of mature
cells exposed to taxifolin at 0.2–20 µg/mL concentrations forthree hours was 107.9 ± 2.2%, respectively. Each experiment
was performed using the non-toxic concentration range oftaxifolin shown in Table 1.

Sakurai M, et al. The Bioactive Flavonoid Taxifolin Inhibits Differentiation and the Production of theInflammatory Cytokine Interleukin-6 in Cultured Human Adipocytes. Adv in Phar & Clin Tria 2022,
7(1): 000195.

Copyright© Sakurai M, et al.

5Advances in Pharmacology and Clinical Trials

Table 1: The viability of adipocytes treated with various taxifolin concentrations.
Test substanceExposure time

Concentration (µg/mL)

Cell viability (%)

Differentiation experiment
Taxifolin

16 d3h

0.2 – 10Anti-inflammatory experiment
0.2 – 20

83.7 ± 3.5

107.9 ± 2.2

The viability of adipocytes treated with various concentrations of taxifolin were measured using CCK-8 assay. Cell viability wascalculated as the percentage of viable cells in the taxifolin-treated group relative to the control group. Data represents the mean
± SEM (n = 18).

Changes in Lipid and Adiponectin During
Differentiation

Figure 1 shows changes in lipid concentrationsin adipocytes and adiponectin in the medium during
differentiation. Photographs show that small matureadipocytes gradually increased until day 16 ofdifferentiation, but many hypertrophic adipocytes wereobserved on day 37. The adipocyte lipid concentration
increased linearly with increasing days of differentiation.
Contrastly, adiponectin concentration in the medium
increased rapidly until day 15 of differentiation, but theincreased level almost reached a plateau on day 31 anddecreased rather rapidly on day 37.

Figure 1: Changes in lipid concentrations in adipocytes and adiponectin concentration in the medium during differentiation.
The upper photographs show typical adipocytes stained with Oil Red O on days 0, 7, 16, and 37 of differentiation. The
concentrations of lipid and adiponectine were measured on days 0, 2, 7, 16, 31, and 37. Each value represents the mean ± SEM(n = 6).

Effect of Taxifolin on Adipocyte Differentiation

Lipid and Adiponectin
The effect of taxifolin on adipocyte differentiation wasassessed by culturing confluent preadipocytes for 16 days inthe differentiation medium (control) or medium containing
taxifolin. Typical photographs of control and taxifolin-treatedadipocytes on day 16 of differentiation are shown in Figure
2. Lipids’ concentration in adipocytes and adiponectin in
the medium measured on day 16 as differentiation markers

are also shown in Figures 2A and 2B, respectively. In the 0.2
and 2 µg/mL taxifolin-treated groups, mature adipocytes
were predominantly observed over immature cells, as in
the control group. Conversely, in the 10 µg/mL taxifolintreated group, immature cells were predominantly observed
over mature adipocytes. Concentrations of both Oil Red
O-stained adipocyte lipids and the medium adiponectin weresignificantly decreased by treating with taxifolin (10 µg/mL)
for 16 d compared to controls.

Sakurai M, et al. The Bioactive Flavonoid Taxifolin Inhibits Differentiation and the Production of theInflammatory Cytokine Interleukin-6 in Cultured Human Adipocytes. Adv in Phar & Clin Tria 2022,
7(1): 000195.

Copyright© Sakurai M, et al.

6Advances in Pharmacology and Clinical Trials

Figure 2: Taxifolin effects on adipocyte differentiation. Confluent preadipocytes were cultured for 16 d in the differentiation
medium (control) or medium containing various taxifolin concentrations (final concentrations: 0.2, 2, and 10 µg/mL). The
upper photographs show typical control and taxifolin-treated adipocytes on day 16 of differentiation (Oil Red O staining). The
concentrations of adipocyte lipid (A) and the medium adiponectin (B) were measured on day16 of differentiation. Each data
represents the mean ± SEM (n = 6). **P < 0.01 vs control: statistical significance was assessed using Dunnett’s test following
one-way ANOVA.
Differentiation-related genes
To further investigate the taxifolin effect on adipocyte
differentiation, expression of differentiation-related genes(PPARγ, C/EBPα, adiponectin, CD36, and GLUT4 mRNAs)
was measured on day 16 in an experiment set separately
using the same procedure. The results are shown in Figure
3. Taxifolin (10 µg/mL) significantly inhibited C/EBPαmRNA expression, but not PPARγ mRNA expression. The
mRNA expression of adiponectin (P < 0.001) and GLUT4 (P< 0.001) was also significantly inhibited by taxifolin (2 and/or 10 µg/mL). Taxifolin (10 µg/mL) tended to inhibit CD36mRNA expression, although it was insignificantly different (P= 0.11).

Sakurai M, et al. The Bioactive Flavonoid Taxifolin Inhibits Differentiation and the Production of theInflammatory Cytokine Interleukin-6 in Cultured Human Adipocytes. Adv in Phar & Clin Tria 2022,
7(1): 000195.

Copyright© Sakurai M, et al.

7Advances in Pharmacology and Clinical Trials

Figure 3: Effects of taxifolin on the expression of differentiation-related genes (PPARγ, C/EBPα, adiponectin, CD36, and GLUT4
mRNAs) in adipocyte differentiation. Confluent preadipocytes were cultured for 16 days in the differentiation medium (control)or medium containing various taxifolin concentrations (0.2, 2, and 10 µg/mL). The quantification of each gene expression in
the adipocytes was measured on day 16 using a real-time PCR system. The gene expression level was expressed relative to thecontrol after normalization using GAPDH. Each data represents the mean ± SEM (n = 6). *P < 0.05 vs control, ***P < 0.001 vscontrol: statistical significance was assessed using Dunnett’s test following one-way ANOVA.

Effect of taxifolin on TNF-α-induced increase in
IL-6
The effect of taxifolin on TNF-α-induced increase ininflammatory cytokine IL-6 was assessed by incubating
mature adipocytes on day 16 of differentiation for threehours in the differentiation medium (control) or the medium

containing TNF-α and TNF-α + taxifolin. After incubation, theIL-6 concentration in the medium was measured, and theresults are shown in Figure 4. TNF-α stimulation significantly
increased IL-6 concentration in the medium. Co-treatment
with taxifolin (20 µg/mL) significantly inhibited the TNF-αinduced increase in IL-6 level.

Sakurai M, et al. The Bioactive Flavonoid Taxifolin Inhibits Differentiation and the Production of theInflammatory Cytokine Interleukin-6 in Cultured Human Adipocytes. Adv in Phar & Clin Tria 2022,
7(1): 000195.

Copyright© Sakurai M, et al.

8Advances in Pharmacology and Clinical Trials

Figure 4: The inhibitory effect of taxifolin on TNF-α-induced increase in inflammatory cytokine IL-6. Mature adipocytes on day
15 of differentiation were incubated for three hours in the differentiation medium (control) or the medium containing TNF-α(final concentrations: 5 ng/mL), TNF-α + various concentrations of taxifolin (final concentrations: 0.2, 2, and 20 µg/mL). Afterincubation, the IL-6 concentrations in the medium were measured. Each data represents the mean ± SEM (n = 6). **P < 0.01 vscontrol, †P < 0.05 vs TNF-α: statistical significance was assessed by Dunnett’s test following one-way ANOVA.

Discussion

This study investigated the taxifolin effect on adipocytes
using non-cytotoxic concentrations, and demonstrated thattaxifolin has anti-differentiation and anti-inflammatory
effects in cultured human adipocytes.

Differentiation or maturation of preadipocytes
to adipocytes is characterized by the appearance of
intracellular lipid droplets and the generation of adiponectin[30]. Therefore, in this study the differentiation process wasevaluated by morphological observation and quantificationof intracellular lipid droplets stained with Oil Red O andadiponectin generation as the differentiation markers.
The number of adipocytes containing lipid droplets andthe lipid concentrations increased with increasing days ofdifferentiation. Adiponectin production rapidly increased
until reaching mature adipocytes on the 16th day ofdifferentiation but decreased conversely on the 37th day
when many swollen adipocytes were observed. This result(Figure 1) is consistent with previous reports showingthat adiponectin production is reduced in hypertrophic
adipocytes [15,17]. Therefore, we decided to verify thetaxifolin effect on differentiation through the differentiation
period for 16 d. Intracellular lipid accumulation was
significantly inhibited in adipocytes cultured in a taxifolincontaining differentiation medium than the control medium.
Cell morphological observations showed that the numberof mature cells in the taxifolin-treated group was lower

compared to the control group. Additionally, the adiponectinconcentration, an adipocyte-specific cytokine, in the medium
on day 16 was significantly lower in the taxifolin group than in
the control group. These results induced by treating taxifolin(Figure 2) suggest that taxifolin may suppress preadipocyteto-adipocyte differentiation.

Adipocyte differentiation and adipogenesis are
regulated by transcriptional factors, including CCAAT/
Enhancer-Binding Protein (C/EBP) family members (C/EBPα, C/EBPβ, and C/EBPδ) and PPARγ [24,31]. In early
adipose differentiation, the expression of transcription
factors such as C/EBPβ and C/EBPδ is temporarily increased
to induce differentiation. Both transcription factors increase
the expression of PPARγ and C/EBPα transcription factors,
thereby increasing the expression of adipocyte-specificgenes, including fatty acid transport proteins (CD36 and
FABP4) and adiponectin, as well as GLUT4. Consequently,
intracellular lipid accumulation and adiponectin production
are promoted and differentiated to mature adipocytes[24,31]. Therefore, to further verify the inhibitory effectof taxifolin on adipocyte differentiation, the expressionof differentiation-related genes such as PPARγ, C/EBPα,adiponectin, CD36, and GLUT4 mRNAs was measured on
day 16 of differentiation. Among the two master regulatorsof adipocyte differentiation, taxifolin significantly inhibited
C/EBPα mRNA expression, but not PPARγ mRNA expression.
Taxifolin also significantly inhibited adiponectin and GLUT4
mRNAs and tended to inhibit CD36 mRNA expression

Sakurai M, et al. The Bioactive Flavonoid Taxifolin Inhibits Differentiation and the Production of theInflammatory Cytokine Interleukin-6 in Cultured Human Adipocytes. Adv in Phar & Clin Tria 2022,
7(1): 000195.

Copyright© Sakurai M, et al.

9Advances in Pharmacology and Clinical Trials(Figure 3). These results further showed that taxifolin
suppressed adipocyte differentiation. Although moredetailed investigation may be needed, taxifolin may havesuppressed the coordination between PPARγ and C/EBPα
by significantly reducing C/EBPα mRNA expression. It was
speculated that this imbalance between PPARγ and C/EBPα
may have suppressed mRNA expression of adiponectin,
CD36, and GLUT4, resulting in the suppression of fat
accumulation and adiponectin production. The excessive
numbers of adipocytes and increased size of adipocytes
in adipose tissue contribute to the obesity induction [32].
Theobromine, a cocoa bean-derived methylxanthine,is thought to prevent obesity by suppressing adipocyte
differentiation or adipogenesis by suppressing C/EBPβ
expression [24]. Therefore, the inhibitory effect of taxifolinon adipocyte differentiation may also be an effective strategy
for preventing and treating obesity.
The development of chronic inflammatory is closely
associated with an increase in infiltration of M1 macrophages
into adipose tissue because adipocyte hypertrophy andmacrophage infiltration were observed in the adipose tissueof patients with obesity [33,34]. TNF-α secreted from M1
macrophages that infiltrate adipose tissue activates NF-κB
via TNF receptors expressed on the surface of adipocytes.
Activated NF-κB translocates into the nucleus, binds tothe inflammatory gene promoter region, and ultimately
promotes the expression and secretion of proinflammatory
cytokines, such as IL-6 and MCP-1 [13,14,16]. A thiazolidine
derivative, which is a PPARγ agonist, has been reported tosuppress the NF-κB pathway via adiponectin produced
through the promotion of the PPARγ-adiponectin pathway
and exerts an anti-inflammatory effect [13,14,35].Therefore,
in this study, TNF-α was added to the medium of cultured
mature adipocytes on day 16 of differentiation instead ofmacrophage infiltration to examine taxifolin effect on TNFα-induced inflammatory cytokines. TNF-α stimulation
significantly increased IL-6 concentration in the medium.
Taxifolin significantly suppressed the TNF-α-induced
increase in IL-6 level, suggesting that taxifolin has antiinflammatory effects (Figure 4). Taxifolin has been reported
to be an Adipo2 receptor agonist [36]. Additionally, pine
bark extract containing taxifolin has been reported toinhibit NF-κB activation in macrophages [37] and vascularendothelial cells [38,39]. These findings suggest that the IL-6
inhibitory effect of taxifolin may be due to a direct inhibition
of NF-κB activity and/or an indirect inhibition of NF-κB
activity mediated by the Adipo2 receptors, although future
verification is necessary.
In conclusion, it was demonstrated here that taxifolin
inhibited adipocyte differentiation and exerted antiinflammatory effects in cultured human adipocytes. This
result will be a useful information for elucidating themolecular mechanism of taxifolin for adipocytes in the

future. Furthermore, combined with previous results on
macrophages [29], taxifolin is expected to have the potential
as a therapeutic drug for obesity and metabolic syndrome.

Conclusion

It was first demonstrated in this study that taxifolin had
anti-differentiation and anti-inflammatory effects in cultured
human adipocytes. Taxifolin is expected to have the potential
as a therapeutic drug for obesity and metabolic syndrome.

Acknowledgements

The authors would like to thank Enago (www.enago.jp)
for the English language review.

Conflicting interests

This work was supported in part by a research grantfrom DHQ Co., Ltd (Saitama, Japan).

References1.

Huang PL (2009) A Comprehensive Definition for
Metabolic Syndrome. Dis Model Mech 2(5-6): 231-237.

3.

Flier JS (2004) Obesity Wars: Molecular Progress
Confronts an Expanding Epidemic. Cell 116(2):337-350.

2.

4.
5.
6.
7.
8.
9.

Matsuzawa Y (1997) Pathophysiology and Molecular
Mechanisms of Visceral Fat Syndrome: The JapaneseExperience. Diabetes Metab Rev 13(1): 3-13.
Friedman JM (2000) Obesity in the New Millennium.
Nature 404(6778): 632-634.

Longo M, Zatterale F, Naderi J, Parrillo L, Formisano P,
et al. (2019) Adipose Tissue Dysfunction as Determinantof Obesity-Associated Metabolic Complications. Int J Mol
Sci 20(9): 2358.

Bray GA, Clearfield MB, Fintel DJ, Nelinson DS(2009) Overweight and Obesity: The Pathogenesis of
Cardiometabolic Risk. Clin Cornerstone 9(4): 30-40.

Chawla A, Chawla R, Jaggi S (2016) Microvasular andMacrovascular Complications in Diabetes Mellitus:
Distinct or Continuum? Indian J Endocrinol Metab 20(4):546-551.
Reaven GM (2011) Insulin Resistance: The Link between
Obesity and Cardiovascular Disease. Med Clin North Am
95(5): 875-892.
Westphal SA (2008) Obesity, Abdominal Obesity, andInsulin Resistance. Clin Cornerstone 9(1): 23-29.

Sakurai M, et al. The Bioactive Flavonoid Taxifolin Inhibits Differentiation and the Production of theInflammatory Cytokine Interleukin-6 in Cultured Human Adipocytes. Adv in Phar & Clin Tria 2022,
7(1): 000195.

Copyright© Sakurai M, et al.

10Advances in Pharmacology and Clinical Trials

10. Kadowaki T, Yamauchi T, Kubota N, Hara K, Ueki K, et al.
(2006) Adiponectin and Adiponectin Receptors in Insulin
Resistance, Diabetes, and the Metabolic Syndrome. J Clin
Invest 116(7): 1784-1792.

11. Matsuzawa Y (2006) The Metabolic Syndrome andAdipocytokines. FEBS Lett 580(12): 2917-2921.

12. Christiansen T, Richelsen B, Bruun JM (2005) Monocyte
Chemoattractant Protein-1 is Produced in Isolated
Adipocytes, Associated With Adiposity and Reduced
After Weight Loss in Morbid Obese Subjects. Int J Obes
(Lond) 29(1): 146-150.
13. Suganami T, Nishida J, Ogawa Y (2005) A Paracrine
Loop between Adipocytes and Macrophages Aggravates
Inflammatory Changes: Role of Free Fatty Acids andTumor Necrosis Factor Α. Arterioscler Thromb Vasc Biol
25(10): 2062-2068.

14. Suganami T, Tanimoto Koyama K, Nishida J, Itoh M, Yuan
X, et al. (2007) Role of the Toll-Like Receptor 4/NF-Κb
Pathway in Saturated Fatty Acid-Induced Inflammatory
Changes in the Interaction between Adipocytes andMacrophages. Arterioscler Thromb Vasc Biol 27(1): 8491.
15. Tilg H, Moschen AR (2006) Adipocytokines: Mediators
Linking Adipose Tissue, Inflammation, and Immunity.
Nat Rev Immunol 6(10): 772-783.
16. Zoico E, Garbin U, Olioso D, Mazzali G, Fratta Pasini AM,
et al. (2009) The Effects of Adiponectin on Interleukin-6
and MCP-1 Secretion in Lipopolysaccharide-Treated
3T3-L1 Adipocytes: Role of the NF-Κb Pathway. Int J Mol
Med 24(6): 847-851.

17. Lau DC, Dhillon B, Yan H, Szmitko PE, Verma S (2005)
Adipokines: Molecular Links between Obesity andAtherosclerosis. Am J Physiol Heart Circ Physiol 288(5):2031-2041.
18. Hammarstedt A, Andersson CX, Rotter Sopasakis V,
Smith U (2005) The Effect of PPARγ Ligands on the
Adipose Tissue in Insulin Resistance. Prostaglandins
Leukot Essent Fatty Acids 73(1): 65-75.
19. Okuno A, Tamemoto H, Tobe K, Ueki K, Mori Y, et al.
(1998) Troglitazone Increases the Number of Small
Adipocytes without the Change of White Adipose Tissue
Mass in Obese Zucker Rats. J Clin Invest 101(6): 13541361.

20. Tao C, Sifuentes A, Holland WL (2014) Regulation of
Glucose and Lipid Homeostasis by Adiponectin: Effects
on Hepatocytes, Pancreatic Beta Cells and Adipocytes.

Best Pract Res Clin Endocrinol Metab 28(1): 43-58.

21. Bhat IA, Kabeer SW, Reza MI, Mir RH, Dar MO (2020)
Adiporon: A Novel Insulin Sensitizer in Various
Complications and the Underlying Mechanisms: A
Review. Curr Mol Pharmacol 13(2): 94-107.
22. Okada Iwabu M, Yamauchi T, Iwabu M, Honma T,
Hamagami K, et al. (2013) A Small-Molecule Adipor
Agonist for Type 2 Diabetes and Short Life in Obesity.
Nature 503(7477): 493-499.

23. Fredholm BB, Lindgren E (1984) The Effect ofAlkylxanthines and other Phosphodiesterase Inhibitors
on Adenosine-Receptor Mediated Decrease in Lipolysis
and Cyclic Amp Accumulation in Rat Fat Cells. Acta
Pharmacol Toxicol (Copenh) 54(1): 64-71.
24. Mitani T, Watanabe S, Yoshioka Y, Katayama S, Nakamura
S, et al. (2017) Theobromine Suppresses Adipogenesis
Through Enhancement of CCAAT-Enhancer-Binding
Protein Β Degradation by Adenosine Receptor A1.
Biochim Biophys Acta Mol Cell Res 1864(12): 24382448.
25. Yang P, Xu F, Li HF, Wang Y, Li FC, et al. (2016) Detection of
191 Taxifolin Metabolites and Their Distribution in Rats
Using HPLC-ESI-IT-TOF-Msn. Molecules 21(9): 1209.

26. Sunil C, Xu B (2019) An Insight into the Health-Promoting
Effects of Taxifolin (Dihydroquercetin). Phytochemistry166: 112066.
27. Ding T, Wang S, Zhang X, Zai W, Fan J, et al. (2018) Kidney
Protection Effects of Dihydroquercetin on Diabetic
Nephropathy Through Suppressing ROS and NLRP3Inflammasome. Phytomedicine 41: 45-53.
28. Sun X, Chen RC, Yang ZH, Sun GB, Wang M, et al. (2014)
Taxifolin Prevents Diabetic Cardiomyopathy in Vivoand In Vitro by Inhibition of Oxidative Stress and Cell
Apoptosis. Food Chem Toxicol 63: 221-232.
29. Muramatsu D, Uchiyama H, Kida H, Iwai A (2020) In
Vitro Anti-Inflammatory and Anti-Lipid Accumulation
Properties of Taxifolin-Rich Extract from The Japanese
Larch, Larix Kaempferi. Heliyon 6(12): e05505.
30. Fu Y, Luo N, Klein RL, Garvey WT (2005) Adiponectin
Promotes Adipocyte Differentiation, Insulin Sensitivity,and Lipid Accumulation. J Lipid Res 46(7): 1369-1379.
31. Lefterova MI, Zhang Y, Steger DJ, Schupp M, Schug J, et al.
(2008) Pparγ And C/EBP Factors Orchestrate Adipocyte
Biology Via Adjacent Binding on a Genome-Wide Scale.
Genes Dev 22(21): 2941-2952.

Sakurai M, et al. The Bioactive Flavonoid Taxifolin Inhibits Differentiation and the Production of theInflammatory Cytokine Interleukin-6 in Cultured Human Adipocytes. Adv in Phar & Clin Tria 2022,
7(1): 000195.

Copyright© Sakurai M, et al.

11Advances in Pharmacology and Clinical Trials

32. Gesta S, Tseng YH, Kahn CR (2007) Developmental
Origin of Fat: Tracking Obesity to Its Source. Cell 131(2):242-256.
33. Xu H, Barnes GT, Yang Q, Tan G, Yang D, et al. (2003)
Chronic Inflammation in Fat Plays A Crucial Role in theDevelopment of Obesity-Related Insulin Resistance. J
Clin Invest 112(12): 1821-1830.

34. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel
RL, et al. (2003) Obesity is Associated with Macrophage
Accumulation in Adipose Tissue. J Clin Invest 112(12):1796-1808.
35. Achari AE, Jain SK (2017) Adiponectin, A Therapeutic
Target for Obesity, Diabetes, and Endothelial Dysfunction.
Int J Mol Sci 18(6): 1321.

36. Sun Y, Zang Z, Zhong L, Wu M, Su Q, et al. (2013)
Identification of Adiponectin Receptor Agonist Utilizing

A Fluorescence Polarization Based High Throughput
Assay. PLOS ONE 8(5): e63354.

37. Cho KJ, Yun CH, Yoon DY, Cho YS, Rimbach G, et al. (2000)
Effect of Bioflavonoids Extracted from the Bark of Pinus
Maritima on Proinflammatory Cytokine Interleukin-1
Production in Lipopolysaccharide Stimulated RAW
264.7. Toxicol Appl Pharmacol 168(1): 64-71.

38. Peng Q, Wei Z, Lau BH (2000) Pycnogenol Inhibits Tumor
Necrosis Factor-Α-Induced Nuclear Factor Κb Activation
and Adhesion Molecule Expression in Human VascularEndothelial Cells. Cell Mol Life Sci 57(5): 834-841.
39. Ohkita M, Kiso Y, Matsumura Y (2011) Pharmacology
in Health Foods: Improvement of Vascular Endothelial
Function by French Maritime Pine Bark Extract(Flavangenol). J Pharmacol Sci 115(4): 461-465.

Sakurai M, et al. The Bioactive Flavonoid Taxifolin Inhibits Differentiation and the Production of theInflammatory Cytokine Interleukin-6 in Cultured Human Adipocytes. Adv in Phar & Clin Tria 2022,
7(1): 000195.

Copyright© Sakurai M, et al.