Inclusion complexes of cysteinyl β-cyclodextrin with baicalein restore collagen synthesis in fibroblast cells following ultraviolet exposure

Article abstract of DOI:10.1002/jcb.27687

Baicalein, a bioactive flavonoid, has poor water solubility, thereby limiting its use in a wide range of biological applications. In the present study, we used inclusion complexes of cysteinyl β-cyclodextrin (β-CD) with baicalein to enhance the stability and solubility of baicalein in aqueous solution. We examined the effects of inclusion complexes of cysteinyl β-CD on collagen synthesis following ultraviolet (UV) irradiation, as well as the mechanisms underlying its effects. Our findings demonstrated that baicalein significantly restored collagen synthesis in the UV-exposed human fibroblast Hs68 cells. In addition, synthetic cysteine functionalized β-CDs were found to promote baicalein-induced collagen synthesis. Inclusion complexes of cysteinyl β-CDs with baicalein significantly upregulated the protein expression of type I collagen and activated the transcription of type I, II, and III collagen. Inclusion complexes of cysteinyl β-CDs with baicalein also downregulated matrix metalloproteinase -1 and -3, and α-smooth muscle actin expression. In addition, inclusion complexes of cysteinyl β-CDs with baicalein attenuated the expression of caveolin-1, but this treatment enhanced the UV-induced phosphorylation of Smad in the transforming growth factor-β pathway. These results suggested that the newly synthesized derivative of CD can be used as a complexing agent to enhance the bioavailability of flavonoids such as baicalein, especially in restoring collagen synthesis.

Full text of DOI:10.1002/jcb.27687

Received: 14 April 2018
DOI: 10.1002/jcb.27687
Accepted: 27 August 2018
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R E S E A R C H A R T I C L E
Inclusion complexes of cysteinyl β‐cyclodextrin with
baicalein restore collagen synthesis in fibroblast cells
following ultraviolet exposure
Joomin Lee1
Jiha Sung2
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Eunsook Ahn2
Hwanhee Kim3
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Seon‐Y. Kim2
Eunae Cho4
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Yujeong Shin2
Seunho Jung3,4
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Seunghyun Ahn2
Seyeon Park2
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1Department of Food and Nutrition,
Chosun University, Gwangju, Korea
Abstract
2Department of Applied Chemistry,
Dongduk Women’s University, Seoul,
Korea
3Department of Bioscience and
Biotechnology, Microbial Carbohydrate
Resource Bank (MCRB), Konkuk
University, Seoul, Korea
4Department of Systems Biotechnology,
Center for Biotechnology Research in
UBITA (CBRU), Institute for Ubiquitous
Information Technology and Applications
(UBITA), Konkuk University, Seoul,
Korea
Baicalein, a bioactive flavonoid, has poor water solubility, thereby limiting its use in a
wide range of biological applications. In the present study, we used inclusion
complexes of cysteinyl β‐cyclodextrin (β‐CD) with baicalein to enhance the stability
and solubility of baicalein in aqueous solution. We examined the effects of inclusion
complexes of cysteinyl β‐CD on collagen synthesis following ultraviolet (UV)
irradiation, as well as the mechanisms underlying its effects. Our findings
demonstrated that baicalein significantly restored collagen synthesis in the UV‐
exposed human fibroblast Hs68 cells. In addition, synthetic cysteine functionalized β‐
CDs were found to promote baicalein‐induced collagen synthesis. Inclusion
complexes of cysteinyl β‐CDs with baicalein significantly upregulated the protein
expression of type I collagen and activated the transcription of type I, II, and III
collagen. Inclusion complexes of cysteinyl β‐CDs with baicalein also downregulated
matrix metalloproteinase ‐1 and ‐3, and α‐smooth muscle actin expression. In
addition, inclusion complexes of cysteinyl β‐CDs with baicalein attenuated the
expression of caveolin‐1, but this treatment enhanced the UV‐induced phosphoryla-
tion of Smad in the transforming growth factor‐β pathway. These results suggested
that the newly synthesized derivative of CD can be used as a complexing agent to
enhance the bioavailability of flavonoids such as baicalein, especially in restoring
collagen synthesis.
Correspondence
Seyeon Park, Department of Applied
Chemistry, Dongduk Women’s
University, Seoul 02748, Korea.
Email: sypark21@dongduk.ac.kr
Funding information
National Research Foundation, Grant/
Award Number:
NRF2016R1D1A1B03930171
K E Y W O R D S
baicalein, collagen, cysteinyl β‐cyclodextrin (β‐CD), inclusion complex, ultraviolet‐exposed
fibroblast
1
INTRODUCTION
exposure). Ultraviolet (UV) irradiation is particularly
important extrinsic risk factor.2 Skin aging involves the
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Collagen plays an important role in maintaining the skin
and is largely responsible for its strength and resilience.
Skin aging is inevitably associated with disturbances in
collagen metabolism, such as decreased synthesis and
increased degradation.1 Skin aging is influenced by both
intrinsic (genetics) and extrinsic factors (environmental
degradation of dermal collagen fibrils in the extracellular
matrix by matrix metalloproteinases (MMPs).3 UV
irradiation of the connective tissue in the skin induces
the production of MMP‐1, MMP‐3, and MMP‐9 in the
normal human skin in vivo. This leads to collagen
degradation, especially of type I procollagen (COLI), the
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J Cell Biochem. 2018;1-12.
wileyonlinelibrary.com/journal/jcb
© 2018 Wiley Periodicals, Inc.
1

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LEE ET AL.
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major structural protein component of the human skin.4
UV irradiation also modulates the activity of various
cytokine receptors, growth factors, and the transforming
growth factor‐β (TGF‐β)/Smad signaling pathways re-
lated to collagen synthesis and acts primarily by
inhibiting procollagen expression.4 Previous studies have
demonstrated that increasing the collagen content of the
dermis is an effective way of delaying skin aging.5
However, increasing the dermal thickness and collagen
density do not have a direct biological application.5
Natural products such as polyphenols have been proven
to be beneficial for reducing the risk of developing skin
disease.6-12 For example, epigallocatechin‐3‐gallate has been
shown to exert anti‐inflammatory and antioxidant activities
by scavenging reactive oxygen species (ROS).6,13-15 It was
reported that oral administration of baicalein and wogonin
(10 or 50 mg/kg) twice daily for 14 consecutive days in
hairless mice prevented an increase in skin thickness and
inhibited UVB irradiation‐induced expression of MMP‐9.16
In addition, apigenin was found to induce dermal collagen
synthesis via the Smad2/3 signaling pathway in primary
human dermal fibroblasts.5 Although various flavonoids
exert useful biological activities, some have limited
bioavailability due to low solubility.17 As a result, supra-
molecular complexation of carbohydrates with flavonoids
has been suggested to increase solubility and thus improve
bioavailability.17 In our previous study, we prepared
inclusion complexes of flavonoids with different cyclic
glucans using a suspension method to screen for effective
host molecules for the target flavonoids.
(Tokyo Chemical Industry Co, Ltd, Tokyo, Japan) (5.0 g,
4.4 mmoL) and then mixed with 1‐(p‐toluenesulfonyl)
imidazole (3.9 g, 17.7 mmoL) for 6 hours. After mixing,
β‐CD was dissolved in a NaOH solution for 20 minutes,
and unreacted precipitates were removed by filtration.
Ammonium chloride (6.1 g, 115 mmoL) was added to
terminate the reaction, and the solution was concentrated
to half of its original volume. Tosyl β‐CD was obtained by
precipitation, and the resulting precipitate was washed and
dried. For cysteinylation, tosyl β‐CD (921 mg, 0.7 mmoL) and
L‐cysteine (251 mg, 2.1 mmoL) were dissolved in 40%
triethylamine (TEA) (v/v) aqueous solution (10 mL) at 85°
C with stirring for 2 days under N2 atmosphere. Then,
cysteinyl β‐CD was purified using Diethylaminoethyl
(DEAE)‐Sephadex A‐25 on a Bio‐gel P‐2 column. The
lyophilized product was obtained with a 30% yield from
the initial amount of β‐CD.
2.2
Preparation of cysteinyl
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β‐CD/baicalein complexes
Inclusion complexes of baicalein and cysteinyl β‐CD were
prepared following the previously described methods.23,26
Baicalein (>99%) (Sigma‐Aldrich, St. Louis, MO) was
dissolved in methanol and combined with an aqueous
solution in an appropriate concentration of cysteinyl
β‐CD. For equilibration, the mixture was stirred at 25°C for
72 hours. Methanol was evaporated and lyophilized. The
lyophilized sample was then dissolved in distilled water and
freeze‐dried overnight.
Baicalein (5,6,7‐trihydroxyflavone) is a naturally occurring
flavonoid originally isolated from the roots of Scutellaria
baicalensis and Scutellaria lateriflor. Baicalein has been used
because of its antifibrogenic,18 anti‐inflammatory,19 antitu-
mor,20 antiangiogenesis,21 and antioxidant22 effects. In the
present study, we showed that baicalein can promote
collagen synthesis in UV‐exposed human fibroblast Hs68
cells. Hs68 is a cell line derived from human foreskin and is
derived from primary cells, making it suitable for investigat-
ing the early events involved in UV‐induced skin metabolism
and signaling.22
2.3
Cell culture conditions and cell
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treatment
Human skin fibroblast (Hs68) cells were purchased from the
American Type Culture Collection (Rockville, MD). Cells
were cultured in Dulbecco modified Eagle medium (DMEM)
(Gibco, Carlsbad, CA) and supplemented with 10% fetal
bovine serum and penicillin‐streptomycin. For cell treatment,
baicalein and cysteinyl β‐CD/baicalein complexes were
dissolved in water. For irradiation, the culture medium
was removed, and phosphate‐buffered saline (PBS) was
added to the cells. Then, the cells were irradiated with UVA
(30 J/cm2) and UVB (30 mJ/cm2). After irradiation, the PBS
was removed, and fresh medium was added to the wells.
Dimethyl β‐CD (DM‐β‐CD) was purchased from Sigma‐
Aldrich.
Here, our results demonstrate that inclusion complexes
comprised of synthetic cysteine functionalized β‐cyclodex-
trins (β‐CDs) and baicalein significantly enhanced collagen
restoration by inducing the TGF‐β/Smad signaling pathway.
2
MATERIALS AND METHODS
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2.1
Cysteinyl β‐cyclodextrin synthesis
2.4
Total cell lysate extraction and
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Western blot analysis
Cysteinyl β‐cyclodextrins (β‐CDs) were synthesized as
previously described.24,25 Briefly, mono‐6‐O‐p‐toluenesulfo-
nyl‐β‐CD (tosyl β‐CD) was first synthesized from β‐CD
Cells were harvested and lysed in radioimmunoprecipitation
assay (RIPA) buffer containing 1% NP‐40, 0.5% sodium

LEE ET AL.
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FIGURE 1 Chemical structure of cysteinyl β‐CD (A) and proposed binding mode of the cysteinyl β‐CD /baicalein complex and
chemical structure of baicalein (B). C, The aqueous solubility of cysteinyl β‐CD and baicalein. β‐CD, β‐cyclodextrin

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LEE ET AL.
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FIGURE 2 Effects of baicalein, cysteinyl β‐CD, and inclusion complexes of baicalein/cysteinyl β‐CD on expression of collagen I protein
in UV‐irradiated Hs68 fibroblasts. A, Cells were irradiated with UVA (30 J/cm2) and UVB (30 mJ/cm2) and incubated with vehicle, 10, 20, or
30 µM of baicalein or inclusion complexes of baicalein, or each equivalent amount of cysteinyl β‐CD for 24 hours. Addition of 0.5 nM
TGF‐β was used as a positive control. Cells were lysed and then immunoblotted with antibodies specific for collagen I or β‐actin. Equal
loading in each lane is shown by similar intensities of β‐actin. B, Quantitative analysis was performed using densitometry. Results are shown
as the mean SD of three independent experiments. Asterisks indicate statistically significant differences between treatment and
UV‐irradiated control. *P < 0.05, Student t test compared with the treated group. β‐CD, β‐cyclodextrin; TGF‐β, transforming growth
factor‐β; SD, standard deviation; UV, ultraviolet
deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 10 ng/mL
phenylmethanesulfonyl fluoride, 0.03% aprotinin, and 1 µM
sodium orthovanadate and subsequently incubated on ice for
30 minutes. Protein concentration was measured using a
bicinchoninic acid (BCA) protein assay kit according to the
manufacturer’s protocol (Bio‐Rad, Richmond, CA). Equal
amounts of protein lysates were separated using 12% SDS‐
polyacrylamide gel electrophoresis and were then transferred
onto a nitrocellulose membrane (Schleicher and Schuell,
Keene, NH). The membrane was blocked by incubation with
5% (w/v) non‐fat dry milk in Tris‐buffered saline with 0.1%
Tween‐20 (TBST) at room temperature for 1 hour and
subsequently was incubated with the appropriate antibody at
4°C overnight. Protein expression was measured using
specific primary antibodies targeting Smad2/3, phospho‐
Smad2, β‐actin (Cell Signaling Technology, Beverly, MA),
caveolin‐1, and procollagen 1 (Santa Cruz Biotechnology,
Santa Cruz, CA). After washing with TBST, the membrane
was incubated with the secondary HRP‐conjugated antibody
(Santa Cruz Biotechnology). Bands were detected using
enhanced chemiluminescence (Amersham Biosciences,
Buckinghamshire, UK). Antibeta actin antibody was used
for normalization. Blots were quantified using a Gel Doc
2000 densitometer (Bio‐Rad).
media. After 24 hours of treatment, total RNA was
extracted by homogenizing cells in Trizol reagent
(Invitrogen, Carlsbad, CA). Complementary DNA
(cDNA) was synthesized from total RNA using oligo
(dT) primers (iNtRON, Gyeonggi‐Do, Korea), reverse
transcriptase (iNtRON), and PCR PreMix (Bioneer Corp,
Daejeon, Korea). qRT‐PCR was performed on a real‐time
PCR system (Mx3000P; Stratagene, Tokyo, Japan) using
SYBR PreMix ExTaq (Takara Bio, Shiga, Japan) or Premix
ExTaq (for use with TaqMan probes; Takara Bio). The
following primer sequences (Bionics, Korea) were used
for PCR analyses: GAPDH: 5′‐CAA AAG GGT CAT CAT
TABLE 1 Relative expression of collagen synthesis induced by
baicalein, cysteinyl β‐CD, and inclusion complexes of baicalein/
cysteinyl β‐CD. Each value of baicalein, cysteinyl β‐CD, and
inclusion complex of baicalein/cysteinyl β‐CD represents relative
expression determined from quantitative analysis of Western blot
analysis (Figure 2). The expected value of a mixture was calculated
from Colby’s equation as follows: Expected value in each
concentration (E) = value of baicalein (X)+value of cysteinyl β‐CD
(Y)–XY/100
Concentration, µM
10
20
30
Cysteinyl β‐CD
9.4 16.6 26.3
32.5 51.2 59.2
38.8 59.3 69.9
Baicalein
2.5
RNA isolation and quantitative
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Expected value of mixture
reverse transcription PCR (qRT‐PCR)
Inclusion complexes of cysteinyl β‐CD with 54.9 95.2 93.8
baicalein
Hs68 cells (1 × 106) were treated with baicalein or
cysteinyl β‐CD/baicalein complexes in a serum‐free
Abbreviation: β‐CD, β‐cyclodextrin

LEE ET AL.
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CTC TG‐3′ (forward) and 5′‐CCT GCT TCA CCA CCT
TCT TG‐3′ (reverse); COL1A1: 5′‐TTT GGA TGG TGC
CAA GGG AG‐3′ (forward) and 5′‐CAC CAT CAT TTC
CAC GAG CA‐3′ (reverse); COL1A2: 5′‐GGC CCT CAA
GGT TTC CAA GG‐3′ (forward) and 5′‐CAC CCT GTG
GTC CAA CAA CTC‐3′ (reverse); CO13A1: 5′‐CGC CCT
CCT TAT GGT CAA GG‐3′ (forward) and 5′‐TTC TGA
GGA CCA GTA GGG CA‐3′ (reverse); MMP‐1: 5′‐TGT
GGT GTC TCA CAG CTT CC‐3′ (forward) and 5′‐CGC
TTT TCA ACT TGC CTC CC‐'3 (reverse); MMP‐3:
5′‐GGT TCC GCC TGT CTC AAG AT‐3′ (forward) and
5′‐GGT TCT GGA GGG ACA GGT TC‐3′ (reverse);
TIMP‐1: 5′‐CTT CTG CAA TTC CGA CCT CGT‐3′
(forward) and 5′‐ACG CTG GTA TAA GGT GGT CT
G‐3′ (reverse); and α‐SMA: 5′‐AAA AGA CAG CTA CGT
GGG TGA‐3′ (forward) and 5′‐GCC ATG TTC TAT CGG
GTA CTT C‐3′ (reverse). GAPDH was selected as the
levels without UV activation. The treatment with higher
concentrations of cysteinyl β‐CD alone appeared to
increase COLI levels; however, these results were not
statistically significant. Treatment with various concen-
trations of baicalein significantly elevated COLI protein
expression levels relative to the UV‐exposed controls.
However, the inclusion complexes of cysteinyl β‐CD with
baicalein at concentrations of 10, 20, and 30 μM were
more effective than baicalein alone in upregulating
COL1A1 expression (Figure 2; Table 1). In addition,
another modified type of β‐CD, dimethyl β‐CD (DM‐β‐
CD) was also used but did not have an effect on COL1A1
expression (Figure 3A and 3B).
Because a flavonoid, apigenin, is known to induce
dermal collagen synthesis in primary human dermal
fibroblasts, the effect of baicalein on collagen synthesis
was compared with that of apigenin. As shown in
endogenous control. Data were analyzed using the 2−▵▵C
t
method.27
2.6
Statistical analysis
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Data are presented as mean standard deviation of three
or four independent experiments. In all experiments,
statistically significant differences were determined using
the Student t test (Sigma Plot, La Jolla, CA). P < 0.05 was
considered statistically significant.
3
RESULT
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3.1
Inclusion complexes of cysteinyl
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β‐CD and baicalein‐enhanced collagen
synthesis in UV‐exposed human fibroblast
Hs68 cells
Cysteinyl β‐CD was synthesized, and inclusion complexes
of cysteinyl β‐CD and baicalein were formed. The chemical
structure of cysteinyl β‐CD is shown in Figure 1A, which
was defined using matrix‐assisted laser desorption ioniza-
tion‐time of flight mass spectrometry and NMR spectro-
scopy.28 The complexation mode of cysteinyl β‐CD and
baicalein was determined from NOESY spectrum29, and the
proposed model, as well as the chemical structure of
baicalein are shown in Figure 1B. As the concentration of
cysteinyl β‐CD increased, the aqueous solubility of baicalein
increased compared with baicalein itself (Figure 1C).
As shown in Figure 2A and 2B, UV irradiation
resulted in an 82% reduction in collagen protein levels
in Hs68 cells after 24 hours of treatment. Type I
procollagen 1 (COL1A1), most abundant protein in skin
connective tissue, are reduced in aged skin.4 TGF‐β1 was
used as a positive control and restored UV‐induced
degradation of collagen levels relative to the control
FIGURE 3 Effects of cysteinyl β‐CD and DM‐β‐CD on the
expression of collagen I protein in UV‐irradiated Hs68 fibroblasts. A,
Cells were irradiated with UVA (30 J/cm2) and UVB (30 mJ/cm2) and
incubated with vehicle, 40 µM or 80 µM of cysteinyl β‐CD or
DM‐ β‐CD for 24 hours. Addition of 0.5 nM TGF‐β was used as a
positive control. Cells were lysed and then immunoblotted with
antibodies specific for collagen I or β‐actin. Equal loading in each lane
is shown by similar intensities of β‐actin. B, Quantitative analysis was
performed using densitometry. Results are shown as the mean SD of
three independent experiments. β‐CD, β‐cyclodextrin; DM‐β‐CD,
dimethyl β‐CD; TGF‐β, transforming growth factor‐β; UV, ultraviolet;
SD, standard deviation

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LEE ET AL.
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FIGURE 4 Effects of baicalein and apigenin on expression of collagen I protein in UV‐irradiated Hs68 fibroblasts. A, Cells were
irradiated with UVA (30 J/cm2) and UVB (30 mJ/cm2) and incubated with vehicle, 10, 20, or 50 µM of baicalein or apigenin for 24 hours.
Addition of 0.5 nM TGF‐β was used as a positive control. Cells were lysed and then immunoblotted with antibodies specific for collagen I or
β‐actin. Equal loading in each lane is shown by similar intensities of β‐actin. B, Quantitative analysis was performed using densitometry.
Results are shown as the mean SD of three independent experiments. Asterisks indicate statistically significant differences between
treatment and UV‐irradiated control. *P < 0.05, Student t test compared with the treated group. TGF‐β, transforming growth factor‐β;
UV, ultraviolet; SD, standard deviation
Figure 4A and 4B, baicalein was more effective than
apigenin at the same concentration in stimulating
collagen synthesis.
protein data, the quantitative polymerase chain reac-
tion results showed that both baicalein alone and
cysteinyl β‐CD complexes with baicalein‐induced
mRNA expression of COL1A1, COL1A2, and COL3A1
when compared with the corresponding control ex-
pression levels in cells irradiated with UV for 24 hours
(Figure 5). Moreover, complexation of baicalein with
cysteinyl β‐CD promoted baicalein‐induced collagen
mRNA synthesis.
We also examined the expression levels of other
extracellular matrix (ECM) environment regulators,
such as MMPs and their inhibitor protein, TIMP. UV
exposure increased MMP‐1 and MMP‐3 mRNA synth-
esis, while treatment with baicalein alone or cysteinyl
β‐CD/baicalein complexes reversed UV‐induced MMP‐
1 and MMP‐3 mRNA levels, as shown in Figure 6. We
observed a statistically significant decline in mRNA
expression of α‐smooth muscle actin (α‐SMA) upon
treatment with baicalein or cysteinyl β‐CD/baicalein
complexes, but not TIP‐1 mRNA expression (Figure 6).
α‐SMA is a marker of activated fibroblasts that
promotes pathological collagen deposition and scar
formation during wound healing.5,30 Therefore, our
findings suggest that baicalein alone and inclusion
complexes of cysteinyl β‐CD and baicalein promoted
collagen synthesis without scar formation.
To investigate whether the complex formation of
baicalein with cysteinyl β‐CD exerted synergistic effects
on collagen restoration, the expected efficacy of the
mixture was calculated according to Colby’s equation.30
As shown in Table 1, the measured efficiencies of the
inclusion complexes were consistently higher than the
expected efficiencies of the mixture, thereby, demonstrat-
ing the synergistic effects of cysteinyl β‐CD and baicalein
inclusive complexes.
3.2
Inclusion complexes of cysteinyl
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β‐CD and baicalein upregulated COL1A1,
COL1A2, and COL3A1 expressions in
UV‐exposed human fibroblast Hs68 cells
We performed real time PCR to determine whether
baicalein and the cysteinyl β‐CD complexes with
baicalein could act at the transcriptional level to
induce collagen synthesis. Hs68 cells were incubated
with baicalein (10 and 20 μM) or cysteinyl β‐CD
complexes with baicalein for 24 hours. mRNA was
extracted from cells, and gene expression levels were
analyzed using qRT‐PCR. In accordance with our

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FIGURE 5 Effects of baicalein, cysteinyl β‐CD, and inclusion complexes of baicalein/cysteinyl β‐CD on procollagen gene expression in
UV‐irradiated Hs68 fibroblasts. Cells were irradiated with UVA (30 J/cm2) and UVB (30 mJ/cm2) and incubated with vehicle, 10 µM or
20 µM of baicalein or baicalein/cysteinyl β‐CD complexes, or 40 µM or 80 µM of cysteinyl β‐CD for 24 hours. After incubation, total RNA was
extracted and qRT‐PCR was carried out using COL1A1, COL1A2, and COL3A1 primers as described in the experimental procedures section.
Results are shown as the mean standard deviation of three independent experiments. Asterisks indicate statistically significant differences
between treatment and UV‐irradiated control. *P < 0.05. β‐CD, β‐cyclodextrin; qRT‐PCR, quantitative real time polymerase chain reaction;
UV, ultraviolet
3.3
Baicalein and inclusion complexes
baicalein complexes were more effective in increasing
phosphorylation than baicalein alone.
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of cysteinyl β‐CD and baicalein‐induced
Smad2 phosphorylation
TGF‐β1 stimulation is known to activate mesenchymal
cells, which in turn induces collagen secretion, inhibits
protease production, and promotes the secretion of protease
inhibitors such as TIMPs. Furthermore, TGF‐β1 has been
demonstrated to exert its effects through the TGF‐β1/
Smad3 signal transduction pathway.31-35
To determine whether baicalein and cysteinyl β‐CD/
baicalein complexes induce Smad activation and collagen
expression, Smad phosphorylation was examined via
Western blot analysis. As shown in Figure 7A and 7B,
treatment of Hs68 cells with both baicalein and cysteinyl
β‐CD/baicalein complexes for 5, 10, 30, and 60 minutes
increased Smad2 phosphorylation in a dose‐dependent
manner until 60 minutes of treatment. Cysteinyl β‐CD/
3.4
Caveolin‐1 is downregulated by
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cysteinyl β‐CD and cysteinyl β‐CD/
baicalein complexes
Caveolin is a membrane protein that has been reported
to modulate collagen synthesis and inhibit TGF‐β
signaling by participating in TGF‐β receptor (TβR)
internalization.36 Moreover, a recent study in mice
reported that intradermal injection of randomly methy-
lated β‐cyclodextrin twice per week for 2 months
induced the downregulation of caveolin and upregula-
tion of COLI.37
Exposure of fibroblast cells to UV radiation and
treatment with baicalein did not lead to changes in

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LEE ET AL.
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FIGURE 6 Effects of baicalein, cysteinyl β‐CD, and inclusion complexes of cysteinyl β‐CD/ baicalein on MMP‐1, MMP‐3, α‐SMA, and
TIMP‐1 expression in UV‐irradiated Hs68 fibroblasts. Cells were irradiated with UVA (30 J/cm2) and UVB (30 mJ/cm2) and incubated with
vehicle, 10 µM or 20 µM of baicalein or baicalein/cysteinyl β‐CD complexes, or 40 µM or 80 µM of cysteinyl β‐CD for 24 hours. After
incubation, total RNA was extracted and qRT‐PCR was carried out using MMP‐1, MMP‐3, α‐SMA, and TIMP‐1 primers as described in the
experimental procedures section. Results are shown as the mean standard deviation of three independent experiments. Asterisks indicate
statistically significant differences between treatment and UV‐irradiated control. *P < 0.05. β‐CD, β‐cyclodextrin; MMP, matrix
metalloproteinase; α‐SMA, α‐smooth muscle actin; qRT‐PCR, quantitative real time polymerase chain reaction; UV, ultraviolet
protein levels of caveolin‐1, as shown in Figure 8 (lanes
4, 7, and 10). However, treatment of cells with cysteinyl
β‐CD and cysteinyl β‐CD/baicalein complexes for
24 hours significantly reduced caveolin‐1 protein levels
compared with corresponding cysteinyl β‐CD‐treated
control (Figure 8; lanes 3, 6, and 9). In particular,
caveolin‐1 levels were markedly reduced upon treat-
ment with cysteinyl β‐CD/baicalein complexes at a
concentration of 30 μM and the equivalent amount of
cysteinyl β‐CD alone.
metabolism and elucidated the molecular mechanisms
underlying the effects of cysteinyl β‐CD/baicalein
complexes on collagen synthesis in human fibroblast
Hs68 cells.
Baicalein has low aqueous solubility and therefore low
bioavailability, thereby limiting its use in various
biological applications. Encapsulation of a hydrophobic
guest compound (baicalein) with a hydrophilic host
(cyclic glucans) is one method of enhancing aqueous
solubility. Cyclodextrins (CDs) are macrocyclic oligosac-
charides composed of 6‐, 7‐, and 8‐α‐(1,4)‐linked glycosyl
units per molecule (α‐, β‐, and γ‐cyclodextrin, respec-
tively). The CDs are characterized by a hollow truncated
cone structure.39 β‐CD is linked by α‐1,4 glycosidic bonds
with a hydrophilic external surface and a hydrophobic
inner surface, allowing it to interact with guest mole-
cules.40 Here, we synthesized mono‐6‐cysteinyl‐β‐CD by
reacting mono‐6‐O‐p‐toluenesulfonyl‐β‐CD with L‐cy-
steine in aqueous conditions. The characteristics of
4
DISCUSSION
|
In skin aging, UV radiation regulates ECM degradation
in the epidermal and dermal layers, including down-
regulation of collagen synthesis and upregulation of
MMP family member.38 In this study, we used cysteinyl
β‐CD/baicalein complexes to reverse UV‐induced

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FIGURE 7 Effects of baicalein and inclusion complexes of cysteinyl β‐CD/baicalein on phosphorylation of Smad in UV‐irradiated Hs68
cells. A, Cells were irradiated with UVA (30 J/cm2) and UVB (30 mJ/cm2) and incubated with vehicle, 30 µM of baicalein or baicalein/
cysteinyl β‐CD complexes for 5, 10, 30, or 60 minutes. Protein levels of total and phosphorylated Smad were measured using Western blot
analysis. Cells were lysed and then immunoblotted with antibodies specific for Smad2/3 and phospho‐Smad2. B, Quantitative analysis was
performed using densitometry, and the results are expressed as activity relative to that of the untreated control. Each value of the
phosphorylation level was normalized to the corresponding Smad protein level. Results are shown as the mean SD of three independent
experiments. Asterisks indicate statistically significant differences between treatment groups and UV‐irradiated group without treatment.
*P < 0.05, Student t test compared with the treated group. #P < 0.05, Student t test compared with baicalein and baicalein/cysteinyl β‐CD
complex treatment. β‐CD, β‐cyclodextrin; SD, standard deviation, UV, ultraviolet
cysteinyl β‐CD and baicalein complexes were previously
analyzed using UV‐Vis spectroscopy, nuclear magnetic
resonance (NMR) spectroscopy, scanning electron micro-
scopy, and X‐ray powder diffractometry.28 NMR spectro-
scopy data revealed the complexation mode of the
prepared inclusion complexes, in which the benzyl
moiety penetrates the β‐CD cavity and the chromene
part is located near the cysteine.28
The present study shows that a reduction in collagen
synthesis in UV‐radiated fibroblast cells was restored by
treatment with cysteinyl β‐CD and baicalein complexes.
Although baicalein alone can promote collagen synth-
esis, inclusion complexes of cysteinyl β‐CD with
baicalein showed synergistic effects in promoting
collagen production, thereby highlighting the useful-
ness of novel types of β‐CD. Exposure to solar UV
radiation causes collagen degradation, which in turn
leads to the formation of wrinkles and ultimately, skin
aging. Therefore, cysteinyl β‐CD/baicalein complexes
are promising candidates that can serve as functional
ingredients for the treatment of wrinkles. Moreover,
inclusion complexes of cysteinyl β‐CD and baicalein
reduced the increased MMP‐1 and MMP‐3 expression in
UV‐irradiated cells. MMP‐1 degrades collagen and is
thought to be the major contributor to skin aging.3
Generally, exposure to UV radiation induces the
expression of MMP‐1, ‐3, and ‐9 in the normal human
epidermis in vivo.2 UV radiation also generates free
radicals and related ROS, which is potentially mediated
by the transcriptional upregulation of MMPs.13
A
previous study showed that treatment of human skin
•−
fibroblasts with H2O2 or O2 caused an increase in
mRNA expression of MMP‐1.41 In human keratinocytes
(HaCaT cells), baicalein also significantly inhibited the
H2O2‐induced increase in MMP‐1 mRNA levels.42
Given that baicalein can also act as an antioxidant,43
it is likely that downregulates the MMP‐1 expression by
scavenging ROS.
Our results also show that UV exposure stimulated
the expressions of MMP‐1 and MMP‐3 and inhibited
the expression of TIMP‐1, an MMP inhibitor protein,
and ECM proteins, such as COL1A1 and COL1A2 in

10
LEE ET AL.
|
collagen synthesis without triggering scar formation as
a side effect.
Downregulation of TβR II protein expression is
known to result in the downregulation of COL1A
expression in human skin in vivo, indicating that
COL1A synthesis is mediated by TGF‐β signaling.44,45
In addition, cDNA microarray studies reported that
TGF‐β influences fibroblast ECM‐related gene expres-
sion.31 TGF‐β acts as a multifunctional cytokine
and plays a prominent role in cell proliferation and
differentiation, tissue remodeling and repair, and
control of ECM synthesis and degradation.38 In the
dermis, TGF‐β acts as a positive growth factor by
promoting transcriptional expression of ECM proteins,
including procollagen.38,46 TGF‐β1 stimulation in
mesenchymal cells upregulates ECM proteins, plasmi-
nogen activator inhibitor‐1 (PAI‐1), and protease
inhibitors such as TIMPs and SERPINE1, and also
leads to downregulation of protease production.32-34
The TGF‐β superfamily directly activates the Smad
signaling pathway through cell surface receptors to
regulate the expression of target genes in the nu-
cleus.47,48 In humans, the Smad family comprises eight
protein members (Smad1‐Smad8).47 Upon activation,
TGF‐β1 binds to the TβR type I and II serine/threonine
kinase receptors in the cell membrane. Following
ligand activation, TβRI phosphorylates and activates
ligand‐specific receptor‐activated Smads; in the case of
TGF‐β, SMAD2, and SMAD3 are activated.46,49 Phos-
phorylated Smad2 and Smad3 form complexes with
Smad4 and are translocated to the nucleus, where they
regulate the transcription of target genes.35,46,47 Here,
we showed that Smad2/3 phosphorylation was in-
creased by baicalein and cysteinyl β‐CD/baicalein
complexes, demonstrating that TGF‐β and Smad2/3
are involved in collagen synthesis.
FIGURE 8 Effects of baicalein and inclusion complexes of
cysteinyl β‐CD/baicalein on caveolin protein levels in
UV‐irradiated Hs68 cells. A, Cells were irradiated with UVA (30 J/
cm2) and UVB (30 mJ/cm2) and incubated with vehicle, 10, 20, or
30 µM of baicalein or cysteinyl β‐CD/baicalein inclusion
complexes, or each equivalent amount of cysteinyl β‐CD for
24 hours. The addition of 0.5 nM TGF‐β was used as a control. Cells
were lysed and then immunoblotted with antibodies specific for
caveolin‐1 or β‐actin. B, Quantitative analysis was performed using
densitometry, and the results are expressed as activity relative to
that of the untreated control. Results are shown as the mean SD
of three independent experiments. Asterisks indicate statistically
significant differences between treatment and UV‐irradiated
control. **P < 0.05, *P < 0.01, Student t test compared with the
treated group. β‐CD, β‐cyclodextrin; UV, ultraviolet
Our study revealed that the inclusion complexes of
cysteinyl β‐CDs with baicalein strongly promoted
collagen synthesis at the mRNA and protein levels
after UV exposure when compared to collagen levels
upon treatment with baicalein alone. Inclusion com-
plexes of CDs with natural products such as flavonoids
successfully improve the water solubility and stability
of the target compounds.50 Therefore, inclusion com-
plexes of cysteinyl β‐CDs with baicalein serve as
promising agents for the prevention and treatment of
skin aging.
Hs68 cells. However, treatment of fibroblasts with
baicalein alone or cysteinyl β‐CD/baicalein complexes
produced the opposite effect, implying that baicalein
and cysteinyl β‐CD complexes with baicalein promoted
ECM remodeling.
We investigated whether baicalein and cysteinyl
β‐CDs in complex with baicalein influence α‐SMA
expression. Our results revealed that baicalein and
cysteinyl β‐CDs in complex with baicalein showed a
slight decrease in α‐SMA expression, a marker of a
pathological collagen deposition and scar formation
during wound healing. Thus, baicalein and cysteinyl
β‐CD/baicalein complexes can be effective in inducing
ACKNOWLEDGMENTS
This work was supported by a National Research foundation
of Korea (NRF) grant funded by the Korean Government
Program for Basic Science (NRF2016R1D1A1B03930171).

LEE ET AL.
11
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ORCID
Seyeon Park
http://orcid.org/0000-0002-2471-3174
isolated from Scutellaria baicalensis roots on skin damage in
acute UVB‐irradiated hairless mice. Eur
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How to cite this article: Lee J, Ahn E, Kim S-Y,
et al. Inclusion complexes of cysteinyl
β‐cyclodextrin with baicalein restore collagen
synthesis in fibroblast cells following ultraviolet
exposure. J Cell Biochem. 2018;1‐12.
https://doi.org/10.1002/jcb.27687