International Journal of

Molecular Sciences

Article
Protective Effects of Ethanol Extract of Brazilian Green Propolis
and Apigenin against Weak Ultraviolet Ray-B-Induced Barrier
Dysfunction via Suppressing Nitric Oxide Production and
Mislocalization of Claudin-1 in HaCaT Cells
Yuta Yoshino 1 , Kana Marunaka 1 , Mao Kobayashi 1 , Haruka Matsunaga 1 , Shokoku Shu 1 , Toshiyuki Matsunaga 2
and Akira Ikari 1, *

1 Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University,

Gifu 501-1196, Japan; yoshino-yu@gifu-pu.ac.jp (Y.Y.); 136033@gifu-pu.ac.jp (K.M.);
155032@gifu-pu.ac.jp (M.K.); 165077@gifu-pu.ac.jp (H.M.); 165041@gifu-pu.ac.jp (S.S.)
2 Education Center of Green Pharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 502-8585, Japan;
matsunagat@gifu-pu.ac.jp
* Correspondence: ikari@gifu-pu.ac.jp; Tel./Fax: +81-(58)-2308124






Citation: Yoshino, Y.; Marunaka, K.;
Kobayashi, M.; Matsunaga, H.; Shu,
S.; Matsunaga, T.; Ikari, A. Protective
Effects of Ethanol Extract of Brazilian
Green Propolis and Apigenin against
Weak Ultraviolet Ray-B-Induced
Barrier Dysfunction via Suppressing
Nitric Oxide Production and
Mislocalization of Claudin-1 in
HaCaT Cells. Int. J. Mol. Sci. 2021, 22,
10326. https://doi.org/10.3390/
ijms221910326
Academic Editor: Michal Zmijewski
Abstract: Once weak ultraviolet ray-B (UVB) irradiates the skin cells, the generation of reactive
nitrogen species (RNS), but not reactive oxygen species (ROS), is stimulated for the mislocalization of
claudin-1 (CLDN1), an essential protein for forming tight junctions (TJs). Since our skin is constantly
exposed to sunlight throughout our lives, an effective protection strategy is needed to maintain the
skin barrier against weak UVB. In the present study, we investigated whether an ethanol extract of
Brazilian green propolis (EBGP) and flavonoids had a protective effect against weak UVB irradiation-
induced barrier dysfunction in human keratinocyte-derived HaCaT cells. A pretreatment with
EBGP suppressed TJ permeability, RNS production, and the nitration level of CLDN1 in the weak
UVB-exposed cells. Among the propolis components, apigenin and apigenin-like flavonoids have
potent protective effects against NO production and the mislocalization of CLDN1 induced by UVB.
The analyses between structures and biological function revealed that the chemically and structurally
characteristic flavonoids with a hydroxyl group at the 40 position on the B-ring might contribute to its
protective effect on barrier dysfunction caused by weak UVB irradiation. In conclusion, EBGP and its
component apigenin protect HaCaT cells from weak UVB irradiation-induced TJ barrier dysfunction
mediated by suppressing NO production.

Keywords: claudin; keratinocyte; UVB; nitric oxide; apigenin; flavonoid

Received: 31 August 2021
Accepted: 23 September 2021
Published: 25 September 2021

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1. Introduction
As part of the skin’s barrier function, tight junctions (TJs) play a critical role in
maintaining the homeostasis of electrolyte ions and water in the tissue surface. The
TJ barrier strand is composed of transmembrane proteins, including claudin (CLDNs)
and occludin, scaffold proteins, zonula-occludens, and cytoskeletal actin, which tightly
close the cell−cell contact area [1]. Since these reports indicate that the expression level
of CLDN1 decreased in the skin with atopic dermatitis [2] and CLDN1 knockout mice
showed skin barrier dysfunction [3], CLDN1 is considered as a main component for the
TJ barriers in the skin among over 20 subtypes of CLDNs. Our recent studies suggest the
expression level and the cellular localization of CLDN1 might be related closely to the
integrity of the TJ barrier in the skin [4,5].
A severe level of ultraviolet ray-B (UVB) irradiation causes reactive oxygen species
(ROS), reactive nitrogen species (RNS), and DNA damage, resulting in the dysfunction
of the skin barrier and an elevation of risk factors for skin cancer [6]. In contrast, non-
cytotoxic “weak” UVB irradiation stimulates RNS generation, but not ROS, leading to

Int. J. Mol. Sci. 2021, 22, 10326. https://doi.org/10.3390/ijms221910326 https://www.mdpi.com/journal/ijms

 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 12

the skin barrier and an elevation of risk factors for skin cancer [6]. In contrast, non-cyto-
Int. J. Mol. Sci. 2021, 22, 10326 toxic “weak” UVB irradiation stimulates RNS generation, but not ROS, leading to barrier 2 of 13
dysfunction associated with the mislocalization of CLDN1 in our previous report [5].
Since our skin is constantly exposed to sunlight throughout our lives, an effective protec-
barrier dysfunction
tion strategy associated
is needed with the
to maintain mislocalization
skin health against ofweak
CLDN1 UVB.in our previous
However, report
there are[5].
no
Since our skin
effective is constantly
dietary supplements exposed to sunlight
to protect the skinthroughout our lives, an barrier
against RNS-related effective protection
dysfunction.
strategy
Therefore, is needed
effectivetocompounds
maintain skin health against
or natural ingredientsweakthat
UVB. However,
constantly therethe
protect are skin
no
effective dietary supplements
from UVB-induced stresses mayto protect
becomethe skin ingredients
useful against RNS-related barrier
for skin care dysfunction.
products.
Therefore, effective
Propolis compounds
is a resinous beeor natural composed
product, ingredientsofthat constantly
floral bud and protect
jelly, the skinhas
which fromdi-
UVB-induced stresses may become useful ingredients for skin care products.
verse and potent biological activities [7]. In recent decades, much research has shown that
Propolisproducts
the natural is a resinous bee product,
derived composed
from propolis haveofpotential
floral budbenefits
and jelly,
forwhich has diverse
the skin, such as
and
protective effect against UV-induced skin damage or sunburn by potent antioxidativethat
potent biological activities [7]. In recent decades, much research has shown and
the natural products derived from propolis have potential benefits for
anti-inflammatory effects [8,9]. Therefore, propolis products are attracting much attention the skin, such as
protective effect against UV-induced skin damage or sunburn by potent
as useful candidates for skin health care. Among the propolis products, an ethanol extract antioxidative and
anti-inflammatory effects [8,9].
of Brazilian green propolis Therefore,
(EBGP), whichpropolis
containsproducts are attracting
various kinds much
of bioactive attention
compounds,
as useful candidates for skin health care. Among the propolis products,
such as flavonoids, phenolic acids, esters, terpenoids, and cinnamic acid derivatives, an ethanol extractis
of Brazilian green propolis (EBGP), which contains various kinds of bioactive compounds,
well known to have multiple physiological functions including antibacterial, anti-inflam-
such as flavonoids, phenolic acids, esters, terpenoids, and cinnamic acid derivatives, is well
mation, antioxidant, and antitumor effects [10–13]. Moreover, we recently reported that
known to have multiple physiological functions including antibacterial, anti-inflammation,
EBGP had a protective effect against oxidative stress-induced skin barrier dysfunction on
antioxidant, and antitumor effects [10–13]. Moreover, we recently reported that EBGP had
human keratinocyte-derived cells via its potent antioxidative effect [4], suggesting the
a protective effect against oxidative stress-induced skin barrier dysfunction on human
utility of the ingredient for skin health. However, it remains unclear whether EBGP has a
keratinocyte-derived cells via its potent antioxidative effect [4], suggesting the utility of
protective effect for the skin barrier without antioxidative activity.
the ingredient for skin health. However, it remains unclear whether EBGP has a protective
In the present study, we investigated whether EBGP and its components had a pro-
effect for the skin barrier without antioxidative activity.
tective effect against weak UVB irradiation-induced barrier dysfunction in human
In the present study, we investigated whether EBGP and its components had a
keratinocyte-derived HaCaT cells. Moreover, the relationship between the structural char-
protective effect against weak UVB irradiation-induced barrier dysfunction in human
acter and the biological functions of each flavonoid and phenylpropanoid were also
keratinocyte-derived HaCaT cells. Moreover, the relationship between the structural
demonstrated.
character and the The present study
biological can of
functions provide useful information
each flavonoid about the structure
and phenylpropanoid and
were also
function of natural
demonstrated. product
The present derivatives,
study can provide leading to information
useful the developmentabout ofthedietary
structuresupple-
and
ments or cosmetic ingredients for skin care in the future.
function of natural product derivatives, leading to the development of dietary supplements
or cosmetic ingredients for skin care in the future.
2. Results
2.2.1.
Results
Protective Effect of EBGP on Weak UVB-Induced Barrier Dysfunction
2.1. Protective Effectreported
We recently of EBGPthaton Weak
weakUVB-Induced
UVB irradiation Barrier Dysfunction
transiently induces mislocalization
We recently
of CLDN1, reported
resulting that
in the weakofUVB
defect irradiation
TJ barrier transiently
function [4]. Theinduces mislocalization
protective of
effect of EBGP
CLDN1, resulting
on barrier functioninwere
the defect of TJ barrier
investigated using function [4]. The protective
the cells cultured effect
on transwell of EBGP
plates. on
Pretreat-
barrier function
ment with EBGP were investigated
significantly using the cells
suppressed weakcultured on transwell
UVB-induced plates.
decrease Pretreatment
in transepithelial
with EBGP significantly suppressed weak UVB-induced decrease in transepithelial
electrical resistance (TER), an indicator of electrolyte ion permeability (Figure 1A). electri-
Fur-
cal resistance (TER), an indicator of electrolyte ion permeability (Figure 1A).
thermore, an increase of lucifer yellow (LY) flux, an indicator of small molecular permea-Furthermore,
an increase
tion, of lucifer yellow
was significantly (LY) flux,
suppressed by an indicator
EBGP of small molecular
in a dose-dependent permeation,
manner (Figurewas
1B).
significantly
These resultssuppressed
suggest that byEBGP
EBGP has
in aprotective
dose-dependent mannerweak
effect against (Figure 1B). These results
UVB-induced TJ bar-
suggest that EBGP has protective effect against weak UVB-induced TJ barrier dysfunction.
rier dysfunction.

Figure1.1.Effect
Figure Effectof
ofEBGP
EBGPononTJ
TJpermeability
permeabilityagainst
againstweak
weakUVB UVBirradiation.
irradiation.The
Thecells
cellswere
werecultured
cultured
ontranswell
on transwell plates
plates and
and incubated
incubated inin the
the absence
absence (vehicle)
(vehicle)and
andpresence
presenceofof5 5oror1010μg/mL
µg/mLEBGP
EBGP for
30 30
for min, followed
min, by exposure
followed to 5tomJ/cm
by exposure
2 UVB
5 mJ/cm for 6 for
2 UVB h. (A)
6 h.TER
(A) was
TERmeasured
was measured by volt-ohm meter.
by volt-ohm
meter. The data were presented as a percentage of control. (B) Paracellular permeability of LY was
measured using a fluorescence spectrometry. n = 4. ** p < 0.01 vs. control. # p < 0.05, ## p < 0.01 and
NS p > 0.05 vs. vehicle.
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 12

The data were presented as a percentage of control. (B) Paracellular permeability of LY was meas-
Int. J. Mol. Sci. 2021, 22, 10326 3 of p13>
ured using a fluorescence spectrometry. n = 4. ** p < 0.01 vs. control. # p < 0.05, ## p < 0.01 and NS
0.05 vs. vehicle.

2.2.
2.2. Protective
Protective Effect
Effect of
of EBGP
EBGP on on Weak
Weak UVB-Induced
UVB-Induced RNS RNS Generations
Generations
Once
Once the weak UVB irradiated the cells, the generation of
the weak UVB irradiated the cells, the generation of reactive
reactive nitrogen
nitrogen species
species
(RNS)
(RNS) was stimulated, which induced mislocalization of CLND1 [5]. Next, the
was stimulated, which induced mislocalization of CLND1 [5]. Next, the effects
effects on
on
RNS 2+
RNS generation
generation were
were investigated
investigated using
using specific
specific probes
probes forforCa
Ca2+ influx,
influx, nitrogen
nitrogen oxide
oxide
(NO)
(NO) production,
production,and andperoxynitrite
peroxynitriteproduction,
production,which
whichis aismore reactive
a more formform
reactive of RNS. The
of RNS.
pretreatment with EBGP significantly suppressed the weak UVB-induced increase in Ca 2+
The pretreatment with EBGP significantly suppressed the weak UVB-induced increase in
influx and production
Ca2+ influx and productionof both NO and
of both NOperoxynitrite (Figure
and peroxynitrite 2). These
(Figure results
2). These suggest
results that
suggest
EBGP has protective effects against weak UVB-induced RNS generations
that EBGP has protective effects against weak UVB-induced RNS generations in the cells. in the cells.

Figure 2.
Figure 2. Effect
Effect of
of EBGP
EBGP on Ca2+
on Ca 2+ influx and RNS generations in the weak UVB-exposed
UVB-exposed cells.
cells. After
After
weak UVB,
exposure to weak UVB, the
the cells
cells were
were cultured
cultured for
for 33 h.
h. The
The cells
cells were
were incubated
incubated with
with 55 µM
μM Fluo-8
Fluo-8
AM, 55 µM
AM, μM DAF-2DA,
DAF-2DA, or or 55 µM
μMNiSPY-3
NiSPY-3forfor30
30min.
min.Nonirradiated
Nonirradiated cells
cells were
wereused
usedas
ascontrol.
control. The
The
fluorescence intensities of Fluo-8, DAF-2, and NiSPY-3 were measured using a microplate
fluorescence intensities of Fluo-8, DAF-2, and NiSPY-3 were measured using a microplate reader. reader. n
= 3–6. ** p < 0.01 vs. control. ## p##< 0.01 and NS p > 0.05 vs. vehicle.
n = 3–6. ** p < 0.01 vs. control. p < 0.01 and NS p > 0.05 vs. vehicle.

In the
In theepidermal
epidermalTJTJ barrier,
barrier, the the cellular
cellular localization
localization and function
and function of CLDNs of CLDNs are
are strictly
strictly regulated by the status of its post-translational modifications [14]. Especially,
regulated by the status of its post-translational modifications [14]. Especially, tyrosine ty-
rosine nitration relates to the mislocalization of CLDN1 in HaCaT cells [5].
nitration relates to the mislocalization of CLDN1 in HaCaT cells [5]. Thus, the level ofThus, the level
of tyrosine-nitrated
tyrosine-nitrated CLDN1
CLDN1 protein
protein waswas evaluated
evaluated by immunoprecipitation
by immunoprecipitation assayassay
usingusing
anti-
anti-nitro-tyrosine
nitro-tyrosine and and anti-CLDN1
anti-CLDN1 antibodies.
antibodies. TheThe pretreatmentwith
pretreatment withEBGP
EBGPsignificantly
significantly
suppressed the UVB-induced elevation of tyrosine-nitrated CLDN1 protein (Figure 3A).
Intracellular NO
Intracellular NO is
is produced
produced from
from L-arginine
L-arginine byby the
the NO
NO synthase
synthase (NOS)
(NOS)family
family[15].
[15]. The
The
expression level of NOS3
NOS3 protein
protein was
was increased
increased by by UVB
UVB irradiation
irradiation and
and suppressed
suppressed by by
pretreatment with EBGP
pretreatment EBGP (Figure
(Figure3B).
3B).These
Theseresults
resultsare
areconsistent
consistentwith
withitsits
suppression
suppressionef-
effects on RNS generation in Figure 2. These results indicate that EBGP suppresses NO
production and RNS-related post-translational modification of CLDN1 protein caused by
weak UVB irradiation.
 fects on RNS generation in Figure 2. These results indicate that EBGP suppr
duction and RNS-related post-translational modification of CLDN1 prot
Int. J. Mol. Sci. 2021, 22, 10326 4 of 13
weak UVB irradiation.

Figure
Figure Effect
3. 3. of EBGP
Effect of EBGPon nitratated-CLDN1 and NOS3 expression
on nitratated-CLDN1 and NOS3 in expression
the weak UVB-exposed
in the weak UV
cells. The cells were incubated in the absence (vehicle) and presence of 10 µg/mL EBGP for 30 min,
The cells were incubated in the absence (vehicle) and presence of 10 μg/mL EBGP
followed by exposure to 5 mJ/cm2 UVB for 6 h. (A) The cell lysates were immunoprecipitated with
lowed by exposure
anti-CLDN1 antibody. The to immunoprecipitants
5 mJ/cm2 UVB forwere 6 h.applied
(A) The cell lysates
to SDS-PAGE andwere immunoprecip
detected using
anti-tyrosine nitration (nitro-tyr) and anti-CLDN1 antibodies. The levels of tyrosine nitration and
CLDN1 antibody. The immunoprecipitants were applied to SDS-PAGE of dete
tyrosine
CLDN1 werenitration (nitro-tyr)
represented and anti-CLDN1
as a percentage antibodies.
of control. n = 3–4. ** p < 0.01The levels ofp tyrosine
vs. control. # < 0.05 nitr
vs. vehicle. (B) The cell lysates were applied to SDS-PAGE and detected using
were represented as a percentage of control. n = 3–4. ** p < 0.01 vs. control. p < 0.0 anti-NOS3 and#
anti-β-actin antibodies. β-actin was used as a loading control. The levels of NOS3 expression were
The cell lysates were applied to SDS-PAGE and detected using anti-NOS3 and anti-
represented as a percentage of control. n = 4. ** p < 0.01 vs. control. # p < 0.05 vs. vehicle.
ies. β-actin was used as a loading control. The levels of NOS3 expression were r
2.3. The Effectsof
percentage of EBGP Components
control. n = 4. **onp RNS
< 0.01Generation
vs. control. # p < 0.05 vs. vehicle.
Polyphenols, cinnamic acid derivatives and flavonoids are well known as biologically
active components of propolis. Since EBGP showed potent protective effects against UVB
2.3. The Effects of EBGP Components on RNS Generation
irradiation-induced RNS generation, we examined the effects of propolis components on
Polyphenols,
UVB-induced cinnamic
RNS generation. Amongacid thederivatives and flavonoids
compounds examined, arewith
the treatment well kno
apigenin completely suppressed weak UVB-induced Ca 2+ flux and production of both
cally active components of propolis. Since EBGP showed potent protective
NO and peroxynitrite (Figure 4A). However, neither caffeic acid phenethyl ester (CAPE),
UVBisirradiation-induced
which a polyphenol, nor artepillinRNS generation,
C, which weacid
is a cinnamic examined
derivative,the effects of pr
suppressed
nents on UVB-induced
UVB-induced RNS generation.RNS Thesegeneration.
results suggestAmong the compounds
that flavonoids examined
might be the main
compound suppressing RNS generation among
with apigenin completely suppressed weak UVB-induced propolis components. A lower dose (1
Ca2+ µM)flux and
2+
of propolis components did not suppress the UVB-induced Ca flux. (Figure 4B). The
both NO
contents and
of the peroxynitrite
compounds in EBGP (Figure
(10 µg/mL) 4A). However,
are less than 1 µMneither caffeic
of compound [16].acid p
(CAPE),the
Therefore, which is a effect
protective polyphenol,
of EBGP mightnor be
artepillin
derived fromC, which is a cinnamic
the combined effects, but acid d
not the effect of a single compound.
pressed UVB-induced RNS generation. These results suggest that flavonoid
main compound suppressing RNS generation among propolis components
(1 μM) of propolis components did not suppress the UVB-induced Ca2+ flu
The contents of the compounds in EBGP (10 μg/mL) are less than 1 μM of c
Therefore, the protective effect of EBGP might be derived from the combin
not the effect of a single compound.
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW
Int. J. Mol. Sci. 2021, 22, 10326 5 of 13

Figure 4. Effects of propolis components on Ca2+ influx and 2+ RNS generations in the weak UVB
Figure 4. Effects of propolis components on Ca influx and RNS generations in th
exposed cells. (A) The cells were incubated in the absence (vehicle) and presence of 10 µM artepillin
posed
C, 10 µM cells.
CAPE, (A) The
and 10 µMcells were
apigenin for incubated in the
30 min, followed absenceto(vehicle)
by exposure andfor
5 mJ/cm2 UVB presence
6 h. of
C, 10 μM CAPE, and 10 μM apigenin for 30 min, followed by exposure
After 3 h, the cells were incubated with 5 µM Fluo-8 AM, 5 µM DAF-2DA, or 5 µM NiSPY-3 for to 5 mJ/c
After
30 3 h,fluorescence
min. The the cells intensities
were incubated with 5and
of Fluo-8, DAF-2, μM Fluo-8
NiSPY-3 AM,
were 5 μMbyDAF-2DA,
measured a microplate or 5 μM
reader. (B) The cells were incubated in the absence (vehicle) and presence
min. The fluorescence intensities of Fluo-8, DAF-2, and NiSPY-3 were of 1 µM artepillin C, 1 measured
µM
CAPE, and 1 µM apigenin. The data were presented as a percentage of control. n = 3–5. ** p < 0.01 vs.
reader. (B) The cells were incubated in the absence (vehicle) and presence of 1 μM a
control. ## p < 0.01 and NS p > 0.05 vs. vehicle.
CAPE, and 1 μM apigenin. The data were presented as a percentage of control. n
2.4.
vs.Chemical
control.Structures andand
## p < 0.01 Biological
NS pActivity in Flavonoids
> 0.05 vs. vehicle.
Since apigenin completely protected cellular barrier function against weak UVB-
induced RNS generation, other flavonoids might also have similar protective effects.
2.4. Chemical Structures and Biological Activity in Flavonoids
Among the flavonoids, we selected those with a structure similarly to apigenin and exam-
Since
ined their apigenin
effects completely
on NO production protected
and CLDN1 cellular barrier
mislocalization. Quercetinfunction
suppressedagainst
both UVB-induced NO production (Figure 5A) and CLDN1 mislocalization (Figure 5B).
duced RNS generation, other flavonoids might also have similar pro
On the other hand, kaempferol and kaempferide tended to suppress NO production, but
Among
not the flavonoids,
significantly. we selected
Kaempferol suppressed those withofaCLDN1.
the mislocalization structure similarly
Chrysin to apige
and apiin,
ined their effects on NO production and CLDN1 mislocalization. Querce
which is a glycoside of apigenin, did not have these effects. These results indicate that
flavonoids might be the main active compounds and apigenin has the strongest effects
both UVB-induced NO production (Figure 5A) and CLDN1 mislocalizati
among the flavonoids. Therefore, the next step is to investigate the effects of apigenin in
On detail.
more the other hand, kaempferol and kaempferide tended to suppress NO p
not significantly. Kaempferol suppressed the mislocalization of CLDN1
apiin, which is a glycoside of apigenin, did not have these effects. These r
that flavonoids might be the main active compounds and apigenin has th
fects among the flavonoids. Therefore, the next step is to investigate the effe
in more detail.
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW
Int. J. Mol. Sci. 2021, 22, 10326 6 of 13

Figure
Figure 5. The
5. effects of apigenin-like
The effects flavonoids onflavonoids
of apigenin-like NO productiononand
NO localization
production of CLDN1
and inlocalizat
the weak UVB exposed cells. (A) After exposing the cells to weak UVB, the cells were incubated
the weak UVB exposed cells. (A) After exposing the cells to weak UVB, the cells w
for 3 h in the absence (vehicle) and presence of 10 µM chrysin, 10 µM quercetin, 10 µM kaempferol,
103µM h in the absence
kaempferide, or 10(vehicle) and
µM apiin. The presence
cells of 10 with
were incubated μM5chrysin,
µM DAF-2DA 10 μMfor 30quercetin,
min. The 10 μM
μM kaempferide,
fluorescence or 10was
intensity of DAF-2 μM apiin.using
measured The acells weremicroplate
fluorescent incubated with
reader. 5 *μM
n = 6. DAF-2DA
p < 0.05
#
vs.fluorescence
control. p < 0.01intensity
and NS p >of DAF-2
0.05 was(B)
vs. vehicle. measured using
Representative a fluorescent
images microplate read
of immunocytochemistry
using anti-CLDN1
vs. control. # pantibody.
< 0.01 andScaleNS
bar p
indicates
> 0.05 10vs.µm.
vehicle. (B) Representative images of immu
using
2.5. anti-CLDN1
Protective antibody.
Effect of Apigenin Scale bar indicates
on UVB-Induced 10 μm.
Barrier Dysfunction
In our previous study, weak UVB irradiation induced the mislocalization of CLDN1,
2.5.
but didProtective Effect
not decrease of Apigenin
the expression levelonofUVB-Induced
the protein [5]. Barrier
Treatment Dysfunction
with apigenin
protected against the UVB irradiation-induced mislocalization of CLDN1 (Figure 6A).
In our
Interestingly, NGprevious study,
-nitro-L-arginine weak
methyl UVB
ester irradiation
(L-NAME), induced
an inhibitor of NOthe mislocaliza
synthesis,
butcompletely
also did not decrease
suppressed the expression level
the mislocalization of the
of CLDN1 protein
caused [5].irradiation
by UVB Treatment wit
(Figure 6A), indicating that intracellular NO production may
tected against the UVB irradiation-induced mislocalization of CLDN1 be a critical response to (Fi
maintain homeostasis of CLDN1 on the TJ area. In the transwell assay, pretreatment with
estingly,
apigenin, but N
not-nitro-L-arginine
G methyl ester
CAPE, suppressed a UVB-induced (L-NAME),
decrease an inhibitor
in TER (Figure 6B) and an of NO
completely
increase suppressed
in the paracellular the
flux of LY mislocalization
(Figure 6C). Moreover, ofthe
CLDN1 caused by UVB irra
level of tyrosine-nitrated
CLDN1 protein was decreased by apigenin (Figure
6A), indicating that intracellular NO production may be 7A). The expression level of NOS3 respon
a critical
protein was decreased by pretreatment with apigenin prior to UVB irradiation (Figure 7B).
homeostasis
These of CLDN1
results suggest on the
that apigenin has aTJ area.protective
potent In the transwell
effect against assay, pretreatment
UVB-induced
but not
barrier CAPE,bysuppressed
dysfunction a UVB-induced
suppressing RNS generation. decrease in TER (Figure 6B) an
the paracellular flux of LY (Figure 6C). Moreover, the level of tyrosine-n
protein was decreased by apigenin (Figure 7A). The expression level of NO
decreased by pretreatment with apigenin prior to UVB irradiation (Figur
sults suggest that apigenin has a potent protective effect against UVB-indu
function by suppressing RNS generation.
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW
Int. J. Mol. Sci. 2021, 22, 10326 7 of 13

Figure
Figure6. Effect of apigenin
6. Effect on TJ permeability
of apigenin and localizationand
on TJ permeability of CLDN1 in the weak
localization ofUVB exposed
CLDN1 in the we
cells. (A) Representative images of immunocytochemistry using anti-CLDN1 antibody. After expo-
cells. (A) Representative images of immunocytochemistry using anti-CLDN1 antib
sure to weak UVB, the cells were cultured for 6 h in the absence (vehicle) and presence of 10 µM
sure to weak UVB, the cells were cultured for 6 h in the absence (vehicle) and pr
apigenin, or 100 µM L-NAME. Scale bar indicates 10 µm. (B,C) After exposure to weak UVB, the cell
apigenin,
cultures or 100plates
on transwell μMwere L-NAME.
incubatedScale
for 6 hbar
withindicates
10 µM CAPE, 10orμm.
10 µM(B,C) After
apigenin. (B) exposure
TER t
cell
was cultures
measured on transwell
by volt-ohm meter. (C)plates werepermeation
Paracellular incubated for
of LY 6 detected
was h withusing
10 μM CAPE, or 10
fluorescence
## p < 0.01 and NS p > 0.05 vs. vehicle.
TER was measured by volt-ohm meter. (C) Paracellular permeation of LY was det
spectrometry. n = 3–4. ** p < 0.01 vs. control.
rescence spectrometry. n = 3–4. ** p < 0.01 vs. control. # p < 0.05, ## p < 0.01 and NS p

Int. J. Mol. Sci. 2021, 22, 10326
cells. (A) Representative images of immunocytochemistry using anti-CLDN1 antibody. A
sure to weak UVB, the cells were cultured for 6 h in the absence (vehicle) and presence
apigenin, or 100 μM L-NAME. Scale bar indicates 10 μm. (B,C) After exposure to weak
cell cultures on transwell plates were incubated for 6 h with 10 μM CAPE, or 10 μM api
TER was measured by volt-ohm meter. (C) Paracellular permeation of LY was 8 ofdetected
13 u
rescence spectrometry. n = 3–4. ** p < 0.01 vs. control. # p < 0.05, ## p < 0.01 and NS p > 0.05 v

Figure
Figure 7. Effect
7. Effect of apigenin
of apigenin on nitration
on nitration of CLDN1ofinCLDN1
the weakin the weak UVB-exposed
UVB-exposed cells. The
cells. The cells were
incubated in the absence (vehicle) and presence of 10 μM apigenin for 30 min, followed by
incubated in the absence (vehicle) and presence of 10 µM apigenin for 30 min, followed by exposure
toto5 mJ/cm 2 2
5 mJ/cmUVB for 6for
UVB h. (A)
6 h.The
(A)cell lysates
The cell were immunoprecipitated
lysates with anti-CLDN1
were immunoprecipitated antibody.
with anti-CLDN1
The immunoprecipitants were applied to SDS-PAGE and detected using anti-tyrosine nitration (nitro-
tyr) and anti-CLDN1 antibodies. The levels of tyrosine nitration of CLDN1 were represented as a
percentage of control. n = 3–5. * p < 0.05 vs. control. # p < 0.05 vs. vehicle. (B) The cell lysates were
applied to SDS-PAGE and detected using anti-NOS3 and anti-β-actin antibodies. β-Actin was used
as a loading control. The levels of NOS3 were represented as a percentage of control. n = 3. * p < 0.05
vs. control. # p < 0.05 vs. vehicle.

3. Discussion
In the present study, the EGBP and some flavonoids showed significant protective
effects against weak UVB irradiation-induced barrier dysfunction by suppressing the
NO-related RNS signal in HaCaT cells. EBGP is composed of various ingredients from
various plants. Since the effects of apigenin were similarly to those of EBGP on UVB-
induced barrier dysfunction, apigenin may be one of active components. We previously
reported that UVB-irradiation is sensed by the opsin-2 protein, leading to transient receptor
potential cation channel subfamily V member 1 (TRPV1) activation-related Ca2+ influx to
cytoplasm [5]. The UVB-induced elevation of intracellular free Ca2+ concentration was
suppressed by both EBGP and apigenin (Figures 2 and 4), suggesting that the UVB-sensed
TRPV1 Ca2+ channel is involved in these protection mechanisms (Figure 8). However, the
detail of the molecular mechanism remains unclear. Further analyses are needed to get
more useful chemical structural information of the flavonoids.
Flavonoids are phenolic compounds isolated from various plants and most of them
have potent antioxidant activity. Many research groups have reported the antioxidant
activity of flavonoids, based on their ability to reduce free radical formation and to scavenge
free radicals [17]. In addition to antioxidant activity, some flavonoids are thought to interact
with specific enzymes or ion channels. The flavonoids of Polygonum orientale L. had an anti-
rheumatoid arthritis effect in a rat model and the interaction of flavonoids with NOS protein
as a target protein was demonstrated in the molecular docking study [18]. Eriodictyol,
a flavonoid, acts as an antagonist of the TRPV1 receptor to induce antinociception [19].
These reports indicate that flavonoids might interact with the NOS protein or TRPV1 Ca2+
channel, up to their specific chemical structural property.

Int. J. Mol. Sci. 2021, 22, 10326
cation channel subfamily V member 1 (TRPV1) activation-related Ca2+ influx to cytoplasm
[5]. The UVB-induced elevation of intracellular free Ca2+ concentration was suppressed by
both EBGP and apigenin (Figures 2 and 4), suggesting that the UVB-sensed TRPV1 Ca2+
channel is involved in these protection mechanisms (Figure 8). However, the detail of the
molecular mechanism remains unclear. Further analyses are needed to get more useful9 of 13
chemical structural information of the flavonoids.

Figure8.8.A
Figure Aproposed
proposedscheme
schemeforforthe
theprotective
protectiveeffect
effectof
ofEBGP
EBGPand
andflavonoids
flavonoidsagainst
against weak
weak UVB-
UVB-
induced barrier dysfunction in HaCaT cells.
induced barrier dysfunction in HaCaT cells.

Flavonoids
The structuralarecharacter
phenolic of compounds
flavonoids, isolated
havingfrommorevarious
than justplants and most
hydroxyl of them
residue on
have potent antioxidant activity. Many research groups have reported
their B-ring position, was reported to relate to their radical scavenging activity [20–22]. the antioxidant ac-
tivity of flavonoids, based on their ability to reduce free radical formation
Similar to these reports, our study revealed that the suppressing effects on weak UVB- and to scavenge
free radicals
induced RNS [17]. In addition
generation were to antioxidant
shown activity,
on the B-ring some flavonoids
hydroxylated are thought
flavonoids (apigeninto inter-
and
act with specific
quercetin), but notenzymes or ion channels.
other flavonoids Thekaempferide
(chrysin, flavonoids ofand Polygonum orientale L. had
apiin). Kaempferol, an
also
aanti-rheumatoid
B-ring hydroxylated arthritis effect insimilarly
flavonoid, a rat model and the
tended interaction
to suppress NO of production.
flavonoids with NOS
Chrysin
protein
does notas a target
have proteingroup
a hydroxyl was demonstrated
on the B-ringincompared
the molecular
with docking
apigenin. study
In the[18]. Eriodic-
structures
tyol, a flavonoid, acts as an antagonist of the TRPV1 receptor to
of kaempferide and apiin, the B-ring hydroxyl residue is methylated and O-glycosylated, induce antinociception
[19]. These reports
respectively. These indicate that flavonoids
results indicate might interact
that the hydroxyl withespecially
residue, at the 40or
the NOS protein TRPV1
position
Cathe
on 2+ channel,
B-ring,up in to their specific
flavonoids maychemical structural
be an essential property.
structure to interact with some target
Thewhich
protein, structural character
has a crucial roleoffor
flavonoids,
RNS signaling having moreUVB-irradiated
in weak than just hydroxyl cells residue on
(Figure 9).
their B-ringapiin,
Moreover, position, was reported to
an O-glycosylated relate
form to their radical
of apigenin, did notscavenging activity [20–22].
show a protective effect
against
Similarweak UVB,
to these suggesting
reports, that the
our study glycoside-formed
revealed flavonoidseffects
that the suppressing mighton notweak
penetrate
UVB-
the cell membrane
induced RNS generationinto the cytosol
were shownbecause
on theofB-ring
their chemical polarity.
hydroxylated However,
flavonoids since
(apigenin
the target proteins of flavonoids in cells are still unknown, further study is needed to
discover what protein is the main target of these flavonoids. Structurally characterized
B-ring-hydroxylated flavonoids might become useful candidates for skin care ingredients
or dietary supplements aimed at protecting skin health from UVB.
Apigenin presented a potent effect on UVB-induced RNS generation (Figure 4). There
are various reports on the effects of apigenin on RNS. In another study, apigenin and
its derivatives also suppressed lipopolysaccharide-induced NO production in mouse
macrophage RAW264.7 cells [23,24]. On the other hand, there are some reports of the
opposite effect of apigenin on NO production. In the endothelial cells, apigenin promotes
NOS activation and NO production [25]. Apigenin promotes antibacterial activity by NO
production [26]. These reports indicate that the effect of apigenin on NO regulation might
be different depending on the tissues or the cell types.
Andrade et al., [16] have reported on the contents of phenolic and flavonoid compounds
in green propolis by ultra-high-performance liquid chromatographic system coupled with
tandem mass spectrometry (artepillin C; 4.80 ± 0.03 mg/g, CAPE; 0.29 ± 0.02 mg/g, apigenin;
0.01 ± 0.00 mg/g). The concentration of EBGP (10 µg/mL) was equal to approximately
0.16 µM of artepillin C, 0.016 µM of CAPE, and 0.0037 µM of apigenin, respectively.
Although the content of flavonoids such as apigenin in EBGP is low, EBGP contains a
wide variety of flavonoids. Thus, the potent protective effect of EBGP might be due to the
combined effect of the flavonoids with characteristic chemical structures.

Int. J. Mol. Sci. 2021, 22, 10326
effect against weak UVB, suggesting that the glycoside-formed flavonoids might not pen-
etrate the cell membrane into the cytosol because of their chemical polarity. However,
since the target proteins of flavonoids in cells are still unknown, further study is needed
to discover what protein is the main target of these flavonoids. Structurally characterized
B-ring-hydroxylated flavonoids might become useful candidates for skin care ingredients
10 of 13
or dietary supplements aimed at protecting skin health from UVB.

Figure9.9.Structural
Figure Structuralcharacters
charactersand
andbiological
biologicalfunction
functionof ofapigenin-like
apigenin-likeflavonoids.
flavonoids.Representative
Representative
chemical structure
chemical structure of
of 5,7-dihydroxylated
5,7-dihydroxylated flavonoid
flavonoid isisshown
shownabove.
above.The
Thegroups atat
groups each position
each on
position
thethe
on B- or
B- C-ring in the
or C-ring in flavonoid are presented.
the flavonoid H, hydrogen;
are presented. OH, hydroxyl
H, hydrogen; group; OCH
OH, hydroxyl 3, methoxy
group; OCH3 ,
group; O-glycone, glycosyl substitution of the hydroxy group. The effects of apigenin and apigenin-
methoxy group; O-glycone, glycosyl substitution of the hydroxy group. The effects of apigenin and
like flavonoids are summarized in the biological functions column.
apigenin-like flavonoids are summarized in the biological functions column.

Apigenin
The presented
function of NO on a potent effectison
skin health UVB-induced
controversial. RNS
The generation
expression (Figure
levels 4). There
of NOS3 and
are various reports on the effects of apigenin on RNS. In another study,
NO production have been reported to be necessary for aryl hydrocarbon receptor-ligand- apigenin and its
derivatives
mediated also suppressed
keratinocyte lipopolysaccharide-induced
differentiation NO production
[27]. In the macrophage, in mouse
NO is required macro-
to possess
phage RAW264.7 cells [23,24]. On the other hand, there are some reports
antibacterial activity for the immune system [26]. In the present study, we demonstrated of the opposite
effect
that anof apigenin on NO
overproduced production.
NO-related signalInmediated
the endothelial cells, apigenin
the UVB-induced malpromotes NOS
effect for the ac-
skin
tivation and NO production [25]. Apigenin promotes antibacterial activity
barrier as RNS. The more detailed functions of NO and NO-related signals in the skin need by NO produc-
tion
to be [26]. These
clarified in areports indicate that the effect of apigenin on NO regulation might be
future study.
different depending
In conclusion, on the
EBGP andtissues or the cell
its component types. protected HaCaT cells from weak UVB
apigenin
Andrade et al.,
irradiation-induced TJ [16] have
barrier reported on
dysfunction, the contents
mediated of phenolic
by suppressing and RNS
NO and flavonoid com-
generation.
pounds in green propolis by ultra-high-performance liquid chromatographic system cou-
4. Materials
pled and Methods
with tandem mass spectrometry (artepillin C; 4.80 ± 0.03 mg/g, CAPE; 0.29 ± 0.02
4.1. Materials
mg/g, apigenin; 0.01 ± 0.00 mg/g). The concentration of EBGP (10 μg/mL) was equal to
approximately 0.16 μM of
Rabbit anti-CLDN1 and artepillin C, 0.016
Alexa Fluor μM of CAPE,
555 anti-rabbit and 0.0037
antibodies wereμM of apigenin,
obtained from
respectively.
Thermo FisherAlthough
Scientificthe
(San content
Diego,ofCA,flavonoids
USA). Goatsuchanti-β-actin
as apigeninantibody
in EBGPwas is low, EBGP
obtained
contains
from Santaa wide
Cruzvariety of flavonoids.
Biotechnology (SantaThus,
Cruz, theCA,
potent protective
USA). L-NAME, effect
LY,ofNiSPY-3,
EBGP might Quest be
due to the combined effect of the flavonoids with characteristic chemical
Fluo-8 AM, rabbit anti-nitrotyrosine, and rabbit anti-NOS3 antibodies were from Do- structures.
jindo Laboratories (Kumamoto, Japan), Biotium (Fremont, CA, USA), Goryo Kagaku
(Hokkaido, Japan), AAT Bioquest (Sunnyvale, CA, USA), R&D Systems (Minneapolis, MN,
USA), and Cell Signaling Technology (Beverly, MA, USA), respectively. DAF-2DA, CAPE,
apigenin, kaempferol, kaempferide, chrysin, quercetin, and apiin were from Cayman
Chemical (Ann Arbor, MI, USA). EBGP was kindly provided from Yamada Bee Company
(Okayama, Japan).

4.2. Cell Cultures

Human skin derived-immortal keratinocyte cell line, HaCaT cells [28] were main-
tained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, Saint Louis, MO, USA)
supplemented with 5% fetal bovine serum (FBS; Sigma-Aldrich, Saint Louis, MO, USA),
0.07 mg/mL penicillin-G potassium, and 0.14 mg/mL streptomycin sulfate in a 5% CO2
atmosphere at 37 ◦ C. The cells were passed using 0.5% trypsin every 3–4 days. EBGP stock
solution (10 mg/mL in 100% ethanol) was dissolved in DMEM to prepare the experimental
medium (5, 10 µg/mL). As the vehicle, 0.1% ethanol containing DMEM was used. In the
Int. J. Mol. Sci. 2021, 22, 10326 11 of 13

other experiments, we used 0.1% dimethyl sulfoxide as the vehicle of propolis components
and the other reagents.

4.3. UVB Irradiation

UVB irradiation was carried out as previously described [5]. Using UV Crosslinker CL-
1000M (Analytik Jena, Upland, CA, USA), UVB peaking wavelength at 302 nm was emitted
on the cells for approximately 7 s at 5 mJ/cm2 . After 6 h from UVB irradiation, the cells
were used for the experiments. Since the cell viability was not affected by 5 mJ/cm2 UVB
irradiation for 24 h in previous study [4], this non-cytotoxic dose was used as “weak” UVB.

4.4. Confocal Microscopy

The cells were cultured on cover glasses in 35 mm dishes (70,000 cells/dish) for 3 days.
After 6 h of UVB irradiation, the cells were fixed with methanol at −30 ◦ C for 15 min.
Then, the cells underwent immunocytochemistry assay using rabbit anti-CLDN1 antibody
(1:100 dilution) and Alexa Fluor 555 secondary antibody. The fluorescence images were
taken near the apical membrane using an LSM700 confocal microscope (Carl Zeiss, Jena,
Germany) as described previously [4].

4.5. Paracellular Permeability Assay

The cells (5,000 cells/well) were cultured on Transwell plates for 3 days (0.4 µm
pore size, Corning, NY, USA). After 6 h of UVB irradiation, TER and the paracellular
permeability to LY, a fluorescent paracellular flux marker, were measured as previously
described [4]

4.6. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Immunoblotting

The cells (70,000 cells/dish) were cultured on 35 mm dishes for 3 days. After 6 h from
UVB irradiation, the cells were collected. In immunoprecipitation assay, we incubated the
cell lysates with anti-CLDN1 antibody and protein G-Sepharose beads for 16 h at 4 ◦ C
with gentle rocking. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) and immunoblotting were performed as previously described [4]. Band density
was quantified using ImageJ software (National Institute of Health software). The signals
were normalized using the β-actin as loading control. In immunoprecipitation assay, the
data were normalized to the signals by CLDN1 as control.

4.7. Intracellular RNS Contents and Free Ca2+ Concentration

The cells (7000 cells/well) were cultured on 96-well plates for 2 days. The cells were
incubated with fluorescent probes for 30 min; DAF-2DA, a fluorescent indicator of NO;
NiSPY-3, a fluorescent peroxynitrite indicator; and Fluo-8 AM, a fluorescent Ca2+ indicator,
respectively. After 3 h of UVB irradiation, the fluorescence intensities were measured using
an Infinite F200 Pro microplate reader (Tecan, Switzerland).

4.8. Statistical Analysis

The data are presented as means ± standard error of the mean. Differences between
groups were analyzed using one-way analysis of variance, and corrections for multiple
comparison were made using Tukey’s multiple comparison test. Comparisons between
two groups were made using Student’s t-test. Statistical analyses were performed using
KaleidaGraph version 4.5.1 software (Synergy Software, Reading, PA, USA). Differences
were assumed as significant at p < 0.05.

Author Contributions: Y.Y., K.M., M.K., H.M., and S.S. performed the experiments and analyzed the
data. T.M. contributed to the experiment plan and to the discussion of the manuscript. Y.Y. and A.I.
contributed to writing the paper. A.I. contributed to supervision of the project, interpretation of the
data. All authors have read and agreed to the published version of the manuscript.
Int. J. Mol. Sci. 2021, 22, 10326 12 of 13

Funding: This work was supported in part by JSPS KAKENHI grant number 19H03373, grant from
Hoyu Science Foundation, Cosmetology Foundation, and Ogawa Science and Technology Foundation
(AI), and collaborative research from Yamada Bee Company, Inc.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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