Effects of the Fruit Extract of Tribulus terrestris on Skin Inflammation in Mice with Oxazolone-Induced Atopic Dermatitis through Regulation of Calcium Channels, Orai-1 and TRPV3, and Mast Cell Activation

Abstract

Ethnopharmacological relevance:
In this study, we investigated the effects of Tribulus terrestris fruit (Leguminosae, Tribuli Fructus, TF) extract on oxazolone-induced atopic dermatitis in mice.

Materials and methods:
TF extract was prepared with 30% ethanol as solvent. The 1% TF extract with or without 0.1% HC was applied to the back skin daily for 24 days.

Results:
1% TF extract with 0.1% HC improved AD symptoms and reduced TEWL and symptom scores in AD mice. 1% TF extract with 0.1% HC inhibited skin inflammation through decrease in inflammatory cells infiltration as well as inhibition of Orai-1 expression in skin tissues. TF extract inhibited Orai-1 activity in Orai-1-STIM1 cooverexpressing HEK293T cells but increased TRPV3 activity in TRPV3-overexpressing HEK293T cells. TF extract decreased β-hexosaminidase release in RBL-2H3 cells.

Conclusions:
The present study demonstrates that the topical application of TF extract improves skin inflammation in AD mice, and the mechanism for this effect appears to be related to the modulation of calcium channels and mast cell activation. This outcome suggests that the combination of TF and steroids could be a more effective and safe approach for AD treatment.

Full text

Research Article
Effects of the Fruit Extract of Tribulus terrestris on
Skin Inflammation in Mice with Oxazolone-Induced Atopic
Dermatitis through Regulation of Calcium Channels,
Orai-1 and TRPV3, and Mast Cell Activation
Seok Yong Kang,1Hyo Won Jung,1Joo Hyun Nam,2Woo Kyung Kim,3
Jong-Seong Kang,4Young-Ho Kim,4Cheong-Weon Cho,4Chong Woon Cho,4
Yong-Ki Park,1and Hyo Sang Bae5
1Department of Herbology, College of Korean Medicine, Dongguk University, 123 Dongdae-ro, Gyeongju 38066, Republic of Korea
2Department of Physiology, College of Medicine, Dongguk University, Dongdae-ro 123, Gyeongju 38066, Republic of Korea
3Department of Internal Medicine, Graduate School of Medicine, Dongguk University, Dongguk-ro 27, Ilsan Dong-gu, Goyang,
Gyeonggi-do 10326, Republic of Korea
4College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon, Republic of Korea
5Department of Sasang Constitutional Medicine, College of Korean Medicine, Dongguk University, 27 Dongguk-ro, Ilsan Dong-gu,
Goyang, Gyeonggi-do 10326, Republic of Korea
Correspondence should be addressed to Yong-Ki Park; yongki@dongguk.ac.kr and Hyo Sang Bae; bjypapa@gmail.com
Received 29 May 2017; Accepted 23 October 2017; Published 14 November 2017
Academic Editor: Vincenzo De Feo
Copyright ©  Seok Yong Kang et al. is is an open access article distributed under the Creative Commons AttributionL icense,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Ethnopharmacological Relevance. In this study, we investigated the eects of Tribulus terrestris fruit (Leguminosae, Tribuli Fructus,
TF) extract on oxazolone-induced atopic dermatitis in mice. Materials and Methods. TF extract was prepared with % ethanol
as solvent. e % TF extract with or without .% HC was applied to the back skin daily for  days. Results.%TFextractwith
.% HC improved AD symptoms and reduced TEWL and symptom scores in AD mice. % TF extract with .% HC inhibited skin
inammation through decrease in inammatory cells inltration as well as inhibition of Orai- expression in skin tissues. TF extract
inhibited Orai- activity in Orai--STIM cooverexpressing HEKT cells but increased TRPV activity in TRPV-overexpressing
HEKT cells. TF extract decreased 𝛽-hexosaminidase release in RBL-H cells. Conclusions. e present study demonstrates
that the topical application of TF extract improves skin inammation in AD mice, and the mechanism for this eect appears to
be related to the modulation of calcium channels and mast cell activation. is outcome suggests that the combination of TF and
steroids could be a more eective and safe approach for AD treatment.
1. Introduction
Atopic dermatitis (AD) is a chronic pruritic inammatory
skin disease caused by abnormal skin barrier function and
aberrant immune responses along with cutaneous hyperre-
activity to environmental triggers []. AD has a complex eti-
ology that involves activation of multiple immunological and
inammatory pathways along with disruption of epidermal
barriers, elevated IgE levels, peripheral eosinophilia, and a
predominance of  cells expressing IL-, IL-, and IL-
 []. e main treatment for AD is skin hydration with
emollients and suppression of cutaneous inammation using
topical steroids to reduce the number and severity of ares
[]. However, the use of steroids should be limited to the
most severe cases due to their side eects, which include
adrenal suppression, osteoporosis, hypertension, diabetes,
obesity,andstriae[].Recently,newtreatmentsforAD,such
as  antagonists, cytokine antagonists, phosphodiesterase
Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2017, Article ID 8312946, 12 pages
https://doi.org/10.1155/2017/8312946

 Evidence-Based Complementary and Alternative Medicine
inhibitors, barrier repair therapies, and allergen-directed
immunotherapy, have emerged. Although these therapeutic
agents have greatly improved patient outcomes, the current
treatments for AD are still not ideal and novel therapeutic
strategies are required in the search for better drugs with
safety and ecacy.
Increasing interest in various medicinal plants and their
bioactive ingredients has led to studies for AD treatment. e
fruits from Tribulu s t errestris L(TribuliFructus:TF,family
Leguminosae) have various uses in traditional medicine,
including pacifying the liver, depression relief, tonify blood
and dispel wind, improving vision, and itch treatment as
well as treating dizziness, insomnia, phlegm nodule, scrofula,
and goiters []. TF is also used in folk medicine for a
tonic, aphrodisiac, palliative, astringent, stomachic, antihy-
pertensive, diuretic, lithontriptic, and urinary anti-infective
agents []. In modern experimental studies, TF has been
reported to have protective eects against liver and kidney
toxicity [–], antidiabetic eects [], and antioxidant eects
[]. is plant contains known bioactive compounds, such
as cytoprotective lignanamides [], anthelminthic [] and
antifungal saponin [], and anti-inammatory N-trans-𝜌-
caeoyl tyramine [].
In traditional Korean medicine (TKM), TF is used for
improving eye trouble, as well as liver and kidney disorders,
cutaneous pruritus, edema, inammation, and tracheitis in
skin diseases []. Furthermore, TF has been prescribed for
the treatment of skin inammation in AD. Nevertheless, the
eects of TF on AD and its mechanism of action require
clarication. In this study, we investigated the eects of TF
extract on skin inammation in an AD animal model and
also investigated the mechanism responsible for the eects
of TF extract in AD on the modulation of calcium channels
and the activation of mast cells. In addition, we evaluated the
possibility of combined treatment with TF extract and steroid
forthedevelopmentofmoreeectiveandsafetherapies.For
this, we conducted an in vivo study through application of TF
extract and/or hydrocortisone in low dose.
2. Materials and Methods
2.1. Plant Materials and Preparation of TF Extract. Dried
fruits of T. terrest r i s (TF) were purchased from the Medicinal
Materials Company (Omniherb, Kyungsan, Korea). TF was
authenticated by Professor Y.-K. Park, a medical botanist in
the Department of Herbology, College of Korean Medicine,
Dongguk University (DUCOM). Voucher specimens were
deposited in the Herbarium of the DUCOM under reg-
istration number -TF-E. TF ( g) were extracted
with  L of % ethanol for h, ltered through Whatman
Grade  lter paper (Sigma-Aldrich, St Louis, MO, USA),
concentrated under a vacuum rotary evaporator at ∘C, and
then lyophilized in a freeze-dryer at −∘CwithmTORR
(Il Shin BioBase Co., Yangju, Korea). e obtained TF power
(yield of .%) was stored at ∘C until use, at which time it
was dissolved in a propylene glycol (Sigma-Aldrich).
2.2. Animals. Experimental -week-old SKH- hairless mice
(20±1g, female) were purchased from Raonbio Co. (Yongin,
Republic of Korea). All mice were maintained under constant
conditions at 23 ± 2∘Cand55 ± 5% humidity with free
access to food and water. Aer acclimation for  week, a h
day/night cycle was maintained. e mice were cared for and
used following the animal welfare guidelines issued by the
Institutional Animal Care and Use Committee (IACUC) at
Dongguk University (IACUC--).
2.3. Preparation of AD Mouse Model. Aer  week of acclima-
tion, the back skin of all animals was shaved using clippers
and  mice were put into each group, including the normal
group, AD-induced control group, % TF extract-applied
group, % TF extract with .% hydrocortisone- (HC-)
applied group, and % HC-applied group as a positive control.
e back skin of all groups, except the normal group, was
treated with  𝜇L of % oxazolone (Ox, Sigma-Aldrich)
dissolved in acetone and olive oil ( : ) on days  and  and
then sensitized with  𝜇Lof.%Oxtwiceadayfromdays
to  (Figure ). During sensitization,  𝜇Lof%TFextract,
% TF plus .% hydrocortisone (HC), or % HC was applied
tothesamepartonceaday.Onday,allmiceweresacriced
and blood and skin tissues were harvested for analysis.
2.4. Evaluation of Transepidermal Water Loss. On day ,
transepidermal water loss (TEWL) was measured in three
dierent parts of the dorsal skin using a TEWL machine
(Vapometer, Deln Technologies Ltd., Finland). ree dier-
ent regions of the dorsal skin were measured for  seconds.
2.5. Evaluation of Symptom Scores. On day , symptom
scores were measured in all mice as follows: , no symptoms;
, mild (dryness, scaling); , moderate (dryness, scaling, and
erosion); , middle (dryness, scaling, erosion, and excoria-
tion); and , severe (dryness, scaling, erosion, excoriation,
and hemorrhage). ree evaluators were blinded and sepa-
rately evaluated the symptoms of each mouse and an average
value for each group was obtained from the three scores.
2.6. Histological Analysis of Skin Tissues. Aer measuring
symptom scores and TWEL, all mice were deeply anes-
thetized with isourane and sacriced and back skin tissues
were isolated. e tissues were xed in % paraformaldehyde,
and paran-formatted tissue blocks were made. Skin sections
were cut into  𝜇m thick sections using a microtome and
stained with hematoxylin and eosin (H&E) as well as tolui-
dine blue. All stained tissues were observed by a microscope
(Leica Co., Wetzlar, Germany). Eosinophils in H&E-stained
tissues and mast cells in toluidine blue-stained tissues were
countedinthreedierentpartsbyblindobservation.
2.7. Immunohistochemistry of Skin Tissues. Skin tissue sec-
tions were deparanized in xylene and dehydrated in graded
alcohol. Aer washing the samples in PBS, sections were
placed in epitope-retrieval buer (DakoCytomation, Car-
penteria, CA, USA) at ∘Cforminandsubsequently

Evidence-Based Complementary and Alternative Medicine 
Sacrice
(in acetone + olive oil) (in acetone + olive oil)
TF or HC treatment
5% Ox 50 ，
12 8 18 24
(Days)
0.5% Ox 60 ，
F : Experimental schematic showing preparation of the AD mouse model.
cooled to room temperature (RT) for an additional  min.
e sections were blocked with % goat serum in PBS,
followed by blocking for endogenous peroxidases stained
with peroxidase block solution (DakoCytomation). Sections
were incubated overnight at ∘Cwithantibodiesagainstanti-
CD and anti-Orai- (Cell Signaling Co., Danvers, USA).
Unbound antibodies were removed the following day by
washing the slides three times with PBS. Areas positive for
CD and Orai- induction were stained brown aer develop-
ment with diaminobenzidine. e slides were counterstained
with ltered Mayer’s hematoxylin (Sigma-Aldrich, St. Louis,
MO), rinsed with distilled water, allowed to dry, and then
mounted for viewing purposes. e images were observed
using a Leica digital camera and microscope (Leica Co.,
Wetzlar, G e rmany ).
2.8. Cell Culture. HEKT cells and RBL-H mast cells
(ATCC, Manassas, VA, USA) were cultured in Dulbecco’s
modied Eagle’s medium (DMEM) containing % fetal
bovine serum (FBS) and % penicillin-streptomycin (Life
Technologies, Carlsbad, CA, USA). For stable transfection
of HEKT cells with TRPV,  𝜇g/mL of blasticidin
(ermo Fisher Scientic, Waltham, MA, USA) was added
for antibiotic selection. HEKT cells were grown at ∘C
in a humidied incubator with % CO2/% O2.RBL-H
cells were grown at ∘Cinahumidied%CO
2incubator.
2.9. hSTIM1 and hOrai-1 Transfection. e cDNAs encod-
ing human Orai- (hOrai-) and human STIM (hSTIM)
were purchased from OriGene Technologies (Rockville, MD,
USA) and then subcloned into pcDNA. according to
the manufacturer’s protocol (Life Technologies). Human
TRPV (pReceiver-M) was purchased from Genecopoeia
(Rockville, MD, USA).
For the electrophysiological experiments, HEKT cells
were seeded in  mm2culture dishes (ermo Fisher
Scientic, Waltham, MA, USA)  day before transfection.
e cells were transfected three times with hSTIM, hOrai-
, and pEGFP-N using Turbofecttransfection reagent
(ermo Fisher Scientic) according to the manufacturer’s
instructions. Transfected cells were selected under a patch
clamp system; that is, cells showing green uorescence due
to expression of green uorescent protein in pEGFP-N were
selected using uorescence microscopy. To record Orai-
currents, hOrai-, hSTIM, and pEGFP-N were transfected
at a ratio of . : . : . Experiments were performed aer  h
of transfection.
2.10. Electrophysiology. Patch pipettes were pulled using
borosilicate thin wall glass capillaries (World Precision
Instruments, Sarasota, FL, USA) in ve stages using a
programmable horizontal Flaming/Brown style micropipette
puller (Model P-; Shutter Instruments, Novato, CA,
USA). Pulled pipette tips were re-polished using a micro-
forger (Narishige, Setagaya, Tokyo, Japan) to .– MΩwhen
they were lled with an internal solution and immersed in
an extracellular solution. Transfected HEKT cells were
transferred into a perfusion chamber (Warner Instruments,
Hamden, CT, USA) mounted on the stage of an inverted
microscope (Nikon, Tokyo, Japan). Current through the cell
membrane was recorded using conventional whole-cell patch
clamp methods. Data were acquired using an Axopatch B
amplier (Molecular Devices, Sunnyvale, CA, USA) and
digitalized using a Digidata A (Molecular Devices) set at
 kHz. To reduce electrical noise, data were ltered through
a low-pass lter at  kHz using pCLAMP . soware
(Molecular Devices). Extracellular solutions were perfused
by a gravity-driven perfusion system at a concentration
of  mL/min. TF extract and chemicals were diluted into
extracellular solution to the desired nal concentrations and
applied to the cell through the perfusion system. Liquid
junction potentials were adjusted to zero before gigaseal
formation. Aer the whole-cell conguration was estab-
lished, cell capacitances were measured and compensated for
electronically using an Axopatch B amplier. To measure
TRPV currents, voltage clamp protocols were applied every
 s from − mV to  mV over  ms. Holding potential
was adjusted to  mV. For hOrai- current measurement,
ramp-like pulses from −mVtomVovermswere
applied every  s at a holding potential of − mV. All voltage
and current trace data were saved on a desktop computer and
analyzed using Clampt ., Prism . (GraphPad, La Jolla,
CA, USA), and Origin . (Microcal, Northampton, MA,
USA). All experiments were performed at room temperature
(–∘C).
2.11. Experimental Solution for Whole-Cell Patch Clamp
Study. To measure TRPV current, a pipette solution was
prepared containing  mM CsCl,  mM EGTA, . mM
CaCl2,  mM MgATP, and  mM HEPES adjusted to pH
. with CsOH. e extracellular solution for TRPV was

 Evidence-Based Complementary and Alternative Medicine
prepared and contained  mM NaCl,  mM KCl,  mM
HEPES,  mM BaCl2,mMMgCl
2,  mM EGTA, and  mM
glucoseadjustedtopH.withNaOH.TomeasureOrai-
current, the pipette solution was prepared with  mM
Cs-glutamate,  mM ,-bis(O-aminophenoxy)ethane-
N,N,N󸀠,N󸀠-tetraacetic acid,  mM MgCl2,mMMgATP,
. mM sodium pyruvate, and  mM HEPES adjusted
to pH ., and the extracellular solution was prepared with
 mM NaCl, . mM KCl,  mM MgCl2,mMCaCl
2,
mMD-glucose,andmMHEPESadjustedtopH..To
activate the Orai- current,  𝜇M inositol ,,,-triphosphate
(IP3), which can deplete endoplasmic reticulum (ER)
Ca2+ stores, was added to the pipette solution before the
experiments.
2.12. 𝛽-Hexosaminidase Release Assay. Acolorimetricassay
was performed to determine 𝛽-hexosaminidase activity in
RBL-H cells using a 𝛽-N-acetyl glucosaminidase (NAG)
activity assay kit (Biovision, Zurich, Switzerland). Cell
culture supernatant ( 𝜇L) was incubated with  𝜇Lof
 mM substrate solution ( mM p-nitrophenyl-N-acetyl-𝛽-
D-glucosaminidase dissolved in . M sodium citrate buer,
pH .) at ∘Cfor.h.eenzymereactionwasterminated
by adding  𝜇L of stop solution (. M Na2CO3/NaHCO3,
pH .), and the absorbance was measured at  nm with a
microplate reader (OASYS, Seoul, Korea).
2.13. HPLC Analysis. Standard solution was prepared by
dissolving a rutin standard in methanol (. mg/mL), and TF
extract was dissolved in methanol ( mg/mL) by sonication
for  min at room temperature. e solution was cen-
trifuged at , rpm for min and then ltered with PVDF
(. 𝜇m). HPLC analysis was performed using a Prominence
HPLC system (Shimadzu, Kyoto, Japan) equipped with two
pumps(LC-AD),anautoinjector(SIL-A),aUV-detector
(SPD-A), and a column oven (CTO-A). An Optimapak
C column ( ×. mm,  𝜇m, RsTech, Korea) was used
at ∘C for separation. e mobile phase was made up with
% glacial acetic acid in water (A) and acetonitrile (B).
All samples were eluted at a . mL/min ow rate, and the
gradient elution conditionswere as follows: % B at baseline,
alinearincreaseto%Bfromtomin,thento%
Bfromtomin,andthenholdingat%Bfrom
to  min. Between the injection of samples, the system was
equilibrated for  min at % B. Detection wavelength was set
at UV  nm. en  𝜇Lofvolumeofallsamplesolutions
was injected using an autoinjector.
2.14. Statistical Analysis. All experimental data were
expressed as mean ±standard deviation (SD) by Graphpad
Prism . (GraphPad Soware, La Jolla, CA, USA).
Comparison of each group was carried out by Student’s
𝑡-test and one-way ANOVA, and 𝑃 < 0.05 was considered to
be statistically signicant.
3. Results
3.1. Eects of TF Extract on AD Skin Symptoms. To investigate
the eects of TF extract on AD skin symptoms, symptom
scores were measured in the dorsal skin of AD mice using
a scoring index. As shown in Figure (a), AD symptoms such
as dryness, scaling, erosion, excoriation, and hemorrhaging
were observed in the dorsal skin of AD control mice and
an application of % TF extract without or with .% HC
improved AD symptoms. Symptom scores were signicantly
elevated in the AD control group (𝑃 < 0.001)comparedto
the normal group. An application of % TF extract with .%
HC on the dorsal skin of AD mice signicantly (𝑃 < 0.001)
reduced symptom scores compared to the control group
(Figure (b)). Application of % HC also signicantly reduced
symptom scores (𝑃 < 0.001), but there was no signicant
dierence in the % TF extract only group.
To investigate the skin moisturizing eects of TF extract,
we next measured TWEL in the dorsal skin of AD mice.
In our results, water loss was signicantly elevated in the
AD control group (𝑃 < 0.001)comparedtothenormal
group (Figure (c)). Application of % TF extract with or
without .% HC in the dorsal skin of AD mice signicantly
(𝑃 < 0.001, respectively) inhibited water loss compared to
the control group. Application of % HC also signicantly
inhibited water loss (𝑃 < 0.001)inADmice.
3.2. Eects of TF Extract on Histological Changes in Skin
Tissues. To investigate the eects of TF extract on skin
inammation, histological changes in dorsal skin tissues were
observed by H&E and toluidine blue staining. H&E staining
conrmed a thicker dermis region along with inltration
of inammatory cells in the oxazolone-induced AD control
group (Figure (a)). Application of % TF extract improved
this histopathological feature and signicantly (𝑃 < 0.001)
reduced the numbers of eosinophils, which were typical
inammatory cells, in the epidermal region (Figure (c)).
Application of % TF extract with .% HC or .% HC alone
also signicantly inhibited inltration of eosinophils. Tolui-
dine blue staining conrmed that mast cells were signicantly
(𝑃 < 0.001)elevatedintheADcontrolandvehiclegroups
(Figure (b)). Application of % TF extract with or without
.% HC signicantly reduced mast cell numbers (𝑃 < 0.05,
resp.), and application of % HC signicantly inhibited mast
cell inltration into skin tissues (Figure (d)).
3.3. Eects of TF Extract on Inltration of CD3+TCells
and Expression of Orai-1 in Skin Tissues. Next, we measured
T cell inltration into skin tissues aer AD induction as
well as expression of Orai- as a major ion channel, which
induces skin barrier dysfunction, using immunohistochem-
istry. Expression of CD+T cells in skin tissues was elevated
along with epidermal thickness in the AD control mice and
was reduced by application of % TF extract with or without
.% HC (Figure (a)). Expression of Orai- was also elevated
in AD mice and reduced by application of % TF extract with
or without .% HC (Figure (b)).

Evidence-Based Complementary and Alternative Medicine 
3.4. Eects of TF Extract on Activation of Orai-1 and TRPV3.
To determine the modulatory eects of TF on ion channel
activation in CD+Tlymphocytes,weperformedawhole-
cell patch clamp study of Orai- calcium channels, which can
generate intracellular calcium signaling via T cell receptor
stimulation to activate T cells (Figure (a)). e Orai- chan-
nel was activated by endoplasmic reticulum (ER) calcium
store depletion via direct coupling with the STIM protein,
which is an ER Ca2+ sensor (Ref). erefore, we cotransfected
Orai- and STIM into HEKT cells. To induce ER Ca2+
store depletion, we added  mM ,-bis(o-aminophenoxy)
ethane-N,N,N󸀠,N󸀠-tetraacetic acid (BAPTA), a strong Ca2+
chelator, and  𝜇MIP
3to the pipette solution. Aer a
membrane break-in, IP3slowly diused to the cytoplasmic
sideandstimulatedtheERIP
3receptor. Eventually, depletion
of ER Ca2+ stores slowly generated an inward rectifying
current, 𝐼Orai-1(Figure (a)(A)). Aer conrming the steady
state 𝐼Orai1(Figure (a)(B)), we perfused  mg/mL of TF
extract (% EtOH) into the bath solution. Treatment with
TF extract signicantly inhibited 𝐼Orai-1by 34 ± 0.09%
compared to control currents (Figure (a)(C)). At the end
of the experiment, we treated an Orai- specic blocker, -
aminoethoxydiphenyl borate (-APB), to conrm its basal
current (Figure (a)(A)).
Next, we investigated whether TF extract also activated
TRPV channels, which are related to skin barrier formation
via transglutaminase activation (Figure (b)). Aer observ-
ing no basal current under control conditions, we added
 mg/mL of TF extract (% EtOH) to the bath solution in
HEKT cells stably expressing TRPV. Aer conrming
steady state 𝐼TRPV3through TF extract treatment, we treated
-APB (>% purity), which is a TRPV agonist, to conrm
maximal current and add  𝜇Mrutheniumred,whichis
an inhibitor of TRPV (Figure (b)(A), (B)). Treatment with
 mg/mL of TF extract signicantly (𝑃 < 0.01)increased
𝐼TRPV3activation to 37 ± 0.12%(− mV) compared to -
APB-treated current (𝐼2-APB ) (Figure (b)(C)). ese results
indicate that TF extract can reduce skin barrier impairment
through activation of TRPV ion channels.
3.5. Eects of TF Extract on Mast Cell Degranulation. To
investigate the inhibitory eects of TF extract on mast
cell degranulation mediated via inhibition of ion channels,
such as Orai- and TRPV, 𝛽-hexosaminidase activity was
measured as a biomarker of degranulation. Release of 𝛽-
hexosaminidase from IgE-antigen (Ag) complex-stimulated
RBL-H mast cells was signicantly elevated compared
to unstimulated cells (Figure (a)). Pretreatment with TF
extract at concentrations of ., ., and . mg/mL sig-
nicantly suppressed degranulation of IgE-Ag complex-
stimulated cells in a dose-dependent manner. In addition,
treatment with TF extract reduced degranulation of RBL-
H cells stimulated with IgE/Ag (Figure (b)). Furthermore,
release of 𝛽-hexosaminidase from IgE/Ag-stimulated RBL-
H cells was signicantly inhibited by treatment with dis-
odium cromoglycate (DSCG), which is a mast cell stabilizer,
and -APB. No cytotoxicity was observed in the MTT assay at
any concentration of TF extract when the cells were incubated
for  h (data not shown). ese results suggest that TF
extract may function as a mast cell stabilizer by inhibiting
IgE-Ag complex-mediated degranulation.
3.6. HPLC Analysis of TF Extract. HPLC analysis was per-
formed to quantify the rutin content in TF extract as a
marker compound. As shown in Figure , rutin appeared at
. min. e calibration curve of rutin in the ranges of . to
. 𝜇g/mL showed a linear regression coecient of ..
e rutin content in the TF extract was .%.
4. Discussion
AD is a chronic inammatory skin disease, and the number
of patients with AD is increasing worldwide []. Topical
steroids are currently the most common AD treatment, and
HC is commonly used to treat skin inammation caused by
numerous conditions, such as allergic reactions, eczema, or
psoriasis []. Although initial AD therapy is typically based
on % HC, new eective therapeutic regimens are needed
due to common side eects, such as skin redness, burning,
itching, peeling, nausea, heartburn, headache, dizziness, and
insomnia.
Herbal therapy for skin disorders has been used in
traditional medicine for thousands of years, and specic
herbs have recently been used as new therapeutic materials
[, ]. TF is an herbal medicine used for the treatment of
cutaneous pruritus, edema, inammation, and tracheitis in
TKM []. In terms of the herbal characteristics, this herb
has a mild temper as well as a pungent and bitter taste. Its
target organ is the liver, and it is eective for the control of
headaches, dizziness, breast lumps, chest pain, ank pain,
intestinal pain, and problems with breast milk circulation.
Modern pharmacological studies have detailed its various
eects against diabetes [] and liver and kidney toxicity []
as well as oxidative damage [], but its herbal features and
clinical ecacy are not well known. erefore, in this study,
we investigated the ecacy of TF extract with or without .%
HC to improve AD using an Ox-induced AD mice compared
to % HC application as a common topical dosage.
AD is a relapsing chronic inammatory disease in the
skin characterized by rash, pruritus, eczema, and xerosis
through abnormal inammatory and hyperimmunological
pathways [, ]. ese pathways include excessive inltra-
tion of inammatory cells, such as macrophages, lympho-
cytes, eosinophils, and mast cells, inltrating skin lesions,
as well as a high level of serum IgE []. e skin barrier
has two key functions, including prevention of excessive
water loss and blockage of harmful substances, such as
irritants and allergens from the environment []. erefore,
disruption of the skin barrier increases TEWL via reduction
of stratum corneum hydration and causes penetration of
harmful substances, which results in opportunistic secondary
infections []. us, this defective inside-outside skin bar-
rier presents typical AD symptoms, such as dryness, scaling,
erosion, excoriation, and hemorrhaging. Accumulation of
inammatory cells in the AD skin barrier is easily observed
and induces production of inammatory cytokines, which

 Evidence-Based Complementary and Alternative Medicine
Nor AD 1% TF 1% TF + 0.1% HC 1% HC
(a)
Nor AD 1% TF TF + 0.1% HC 1% HC
0
1
2
3
A∗∗∗
B∗∗∗
B∗∗∗
B∗
Symptom scores
(b)
Nor AD 1% TF TF + 0.1% HC 1% HC
0
10
20
30 A∗∗∗
B∗∗∗
B∗∗∗
B∗∗∗
TEWL (g/Ｇ2Ｂ)
(c)
F : Eects of TF extract on symptoms and transepidermal water loss in oxazolone-induced AD mice. TF extract and HC dissolved
in propylene glycol were applied to the dorsal skin of oxazolone-induced AD mice once a day from days  to . AD-like symptoms were
examined with the naked eye and representative features were recorded (a). Symptom scores were measured in all mice as follows: , no
symptoms; , mild (dryness, scaling); , moderate (dryness, scaling, and erosion); , middle (dryness, scaling, erosion, and excoriation); and
, severe (dryness, scaling, erosion, excoriation, and hemorrhage) (b). TEWL was measured in three dierent parts of the dorsal skin with a
Vapometer for  seconds (c). e results are expressed as the mean ±SD (𝑛=8per a group). ∗𝑃< 0.05 and ∗∗∗𝑃< 0.001 versus normal (A)
or AD control (B) mice. Nor, normal group; AD, oxazolone-induced AD group; % TF, % TF-applied group in AD control; % TF + .%
HC,%TF,and.%HC-appliedgroupinADcontrol;and%HC,%HC-appliedgroupinADcontrol(𝑛=8per a group).
leads to skin barrier remodeling with increased epidermal
and dermis thickness as well as accumulation of inamma-
tory cytokines. In particular, mast cells play a key role in
AD pathogenesis and are activated by cross-linking of the
high anity IgE receptor (Fc𝜀RI) and B cell-producing IgE,
which results in the release of  cytokines, IL-, and IL-
, which induce phenotypic symptoms, such as the IgM
to IgE switch, brosis, epithelial hyperplasia, and barrier
dysfunction[,].Eosinophilsalsocontributetoskinbarrier
remodeling through production of  cytokines, IL-, and
IL-. In our study, TF extract improved skin symptoms in
Ox-induced AD mice via reduction of TWEL and inhibition
of inammatory cell inltration, such as eosinophils and
mast cells, which suggests that TF extract can protect the
skin barrier against water loss and atopic inammation. We
also conrmed the inhibitory eects of TF extract on mast
cell degranulation by measuring 𝛽-hexosaminidase release
alongwithmorphologicalchanges.eseresultssuggestthat
TF extract application to the skin of AD mice can improve
symptomsthroughregulationofmastcell-mediatedallergic
responses and skin inammation.
Since the introduction of Nc/Nga mice as a spontaneously
occurring model of AD, several other AD mouse models
induced by skin injury and epicutaneous sensitization with
allergens, such as ovalbumin, house dust mites, or hapten,
have been developed over the past two decades [, ].
Ox is a heterocyclic compound and is used for development
of AD model []. When applied to the skin of hairless
mice, the mice developed AD symptoms, such as barrier
dysfunction, secretion of IgE and  cytokines, hyperplasia
of epithelial cells, brosis, and inltration of inammatory
cells into the dermis and epidermis []. In this study, we
observed that skin symptoms in Ox-induced AD mice were
improved by application of % TF extract or a combination
of .% HC in low dose, which was similar to the eects
of % HC, and that improvement suggested that TF extract
could prevent skin barrier destruction. Meanwhile, we did
not observe changes in IgE and  cytokine levels (data not
shown), which suggested that our model included the acute
AD phase. Several studies have reported that an Ox challenge
for  weeks in mice could not induce  and  cytokine
production in serum but did lead to cytokine production in

Evidence-Based Complementary and Alternative Medicine 
AD 1% TF 1% TF + 0.1% HC 1% HCNor
(a)
AD 1% TF 1% TF + 0.1% HC 1% HCNor
(b)
Nor AD 1% TF TF + 0.1% HC 1% HC
0
10
20
30
A∗∗∗
B∗
B∗∗∗
B∗
e numbers of eosinophils (no.)
(c)
Nor AD 1% TF TF + 0.1% HC 1% HC
0
50
100
150
A∗∗∗
B∗
B∗
B∗
Mast cells counts (no.)
(d)
F : Eects of TF extract on histopathological changes in the skin tissues of oxazolone-induced AD mice. Dorsal skin tissues were
stained with H&E (a) or Toluidine blue (b) and observed under a microscope (× for H&E, × for toluidine blue). Eosinophils (c) and
mast cells (d) were counted in epidermal regions. e results are expressed as the mean ±SD (𝑛=8per a group). ∗𝑃< 0.05 and ∗∗∗𝑃< 0.001
versus normal (A) or the AD control (B) group.
ear or spleen tissues [–]. erefore, they recommended
a longer challenge period for in vivo study of the induction of
serum IgE or  cytokines.
Recently, investigations into ion channels, such as Orai-,
TRPV, and TRPV, have shed new light on potential targets
for the treatment of inammatory skin diseases, such as AD.
ese ion channels have been shown to directly modulate
epidermal proliferation, dierentiation, barrier homeostasis,
and inammation in the skin []. Ca2+ inux through
these channels eventually generated intracellular Ca2+ sig-
naling, which resulted in dierent outcomes depending
on the individual Ca2+ channel type, such as lymphocyte
activation through Orai- [], epidermal barrier formation
and keratinocyte dierentiation through TRPV [] and
itch generation through TRPV []. erefore, a specic
agonist/antagonist for each calcium channel is required for
maintenance of skin barrier homeostasis and for treatment
of dermatological diseases, such as AD. erefore, Orai-
 and TRPV could be potential targets for the treatment
ofAD.Recently,asapartofourongoingresearchto
nd ion channel-modulating medicinal plants from natural
sources, we found that the application of % TF extract
with or without .% HC reduced Orai- expression and
inltration of CD+T cell into skin tissues. BTP is a potent
inhibitor of Orai- channel which regulates the activation of
immunocytes such as T lymphocytes. BTP decreases store-
operated Ca+ entry (SOCE) mediated by Orai- channels.
Subsequently, Ca+ dependent functional responses such as
the activation of calcineurin and cytokine production are
suppressed in lymphocytes [, ]. -APB is a common
activator of TRPV channels such as TRPV, TRPV, and
TRPV []. Ruthenium red not only blocks TRPV but also
inhibits TRP channels in a broad-spectrum specically [].
In our in vitro study, treatment with TF extract resulted in
signicant inhibition of Orai- activation and induction of
TRPV activation. ese ndings suggest that TF extract
can reduce skin barrier dysfunction associated with allergic
inammation in AD through modulation of the ion channels
Orai- and TRPV. In our previous study, we found the

 Evidence-Based Complementary and Alternative Medicine
AD 1% TF 1% TF + 0.1% HC 1% HCNor
(a)
AD 1% TF 1% TF + 0.1% HC 1% HCNor
(b)
F : Eects of TF extract on inltration of CD+ T cells and expression of Orai- in the skin tissues of oxazolone-induced AD mice.
Dorsal skin tissues were stained with anti-CD (a) or anti-Orai- (b) antibodies and then observed under a microscope (×). Brown color,
CD+ T cells or Orai--expressing cells.
−80
−60
−40
−20
0
−100 −50 0 50
(1)
(2)
Time (sec)
(1)
(2)
(A) (B) (C)
0.0
0.2
0.4
0.6
0.8
1.0
Normalized current
(1)
(1
(1
(1
(1
(2)
(2)
(2)
(2)
(2)
TF extract 1ＧＡ/Ｇ，
0
−30
−60
−90
−120
I(pA/pF)
I(pA/pF)
V(mV)
0.66 ± 0.09
I／Ｌ；Ｃ-1
0 200 400 600 800 1000
TF extract 1ＧＡ/ Ｇ， BTP210－
At −120 Ｇ６
(a)
−100 −50 0 50 100
−1000
−500
0
500
1000
1500
(1)
(2)
(A)
0 200 400 600
−1000
−500
0
500
1000
1500
Time (sec)
(1)
(2)
(B)
0.0
0.2
0.4
0.6
0.8
1.0
Normalized current
(C)
TF extract 1ＧＡ/Ｇ，
TF extract 1ＧＡ/Ｇ，
I(pA/pF)
I(pA/pF)
V(mV)
∗∗
0.37 ± 0.12
I2-！０＂
R.R 50 －
2-APB 100 － At −100 Ｇ６
(b)
F : Eects of TF extract on calcium channels in HEKT cells. Orai- current (𝐼Orai-1) in HEKT cells coexpressing Orai-/STIM
(a). Representative chart trace recordings of 𝐼Orai-1currents (A). e number of parentheses represents steady state 𝐼Orai-1() and its inhibition
by  mg/mL of TF extract (). Related I-Vrelationships of () and of () in (b). Summary of inhibition rate of 𝐼ORAI1by TF extract at − mV
(C). e results are expressed as the mean ±SD (𝑛=3). ∗𝑃< 0.05 versus 𝐼Orai-1. Eects of TF extract on activation of TRPV in HEKT
cells overexpressing TRPV (b). Representative chart recording of 𝐼TRPV3activation by TF extract (A). e number of parentheses represents
steady state TRPV current (𝐼TRPV3)aerTFextracttreatment()andthemaximalcurrentinducedby𝜇M-APB(𝐼2-APB)().Related
current-voltage (I-V) relationship curve at () and () from Figure (b). Normalized graph of 𝐼TRPV3activation by TF extract and -APB at
−mV(C).eresultsareexpressedasthemean±SD (𝑛=3). ∗∗𝑃< 0.01 versus 𝐼2-APB.

Evidence-Based Complementary and Alternative Medicine 
0
10
20
30
TF extract (mg/mL) 0 0 0.1 0.2 0.5 0 0
DNP-IgE+HSA + + + + + +
DSCG (M/mL) 30
2-APB (M/mL)
−
−−−−− −
−−−−−−
100
A∗∗
B∗
B∗B∗
NAG activity (mU/mL)
(a)
NC
TF0.2 TF0.5 2-APB
DSCG
(b)
F : Eects of TF extract on 𝛽-hexosaminidase activity and degranulation in IgE/Ag-stimulated RBL-H cells. e cells were stimulated
with anti-DNP-IgE (. 𝜇g/mL) for  hr, treated with TF extract (., ., and . mg/mL), DSCG ( 𝜇M/mL), or -APB ( 𝜇M/mL) for
hr,andthenstimulatedwithDNP-HAS(𝜇g/mL) for  hr. 𝛽-Hexosaminidase activity was measured in culture supernatants using an NAG
activity assay kit (a). All data are expressed as the mean ±SD (𝑛=3). ∗𝑃< 0.05 and ∗∗𝑃< 0.01 versus normal (A) or DNP-IgE control (B)
cells. e cells were stained with toluidine blue and morphological changes were observed under a microscope (×) (b). e arrows indicate
the degranulated cells. N, normal cells; C, DNP-IgE+HSA-stimulated cells; DSCG, DSCG-treated cells; TF., TF extract (. mg/mL)-treated
cells; TF., TF extract (. mg/mL)-treated cells; and -APB, -APB-treated cells.
regulatory eects of TF methanol extract on Orai- and
TRPV calcium channels [].
We detected rutin as a main compound in TF extract
via HPLC analysis. e suppressive eects of rutin on AD
and allergic contact dermatitis have been reported [].
However, further studies are required to investigate the
recovery mechanism of TF extract in skin barrier dysfunction
through modulation of calcium channels to understand the
therapeutic eects of TF extract and rutin on AD. Recently,
the genotoxic eect of TF extract at the high concentrations
in cultured peripheral human lymphocytes has been reported
[]. Although in vitro study, this should be taken into
consideration for the development of new herbal medicines
using TF extract.

 Evidence-Based Complementary and Alternative Medicine
(1)
2.0
1.5
1.0
0.5
0.0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0
(min)
(a)
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0
(min)
(1)
(b)
F : HPLC chromatogram of TF extract (a) and rutin (b) standard analyzed on an Optimapak C column with a gradient elution of
mobile phases of % glacial acetic acid in water and acetonitrile along with wavelength detection at UV  nm. Peak identication: (1) rutin.
5. Conclusions
We observed that application of % TF extract or a combi-
nation of TF extract with .% HC to the skin of oxazolone-
induced AD had benecial eects on skin barrier function,
had been protected against excessive water loss, and had
inhibited inammation by blocking inltration of inam-
matory cells, such as T cells and eosinophils. e working
mechanisms aect modulation of the calcium channels Orai-
 and TRPV, as well as inhibiting mast cell activation. is
result suggests that TF extract can be used as a natural source
for the development of new medicines to reduce toxicity
induced by topical steroids and increase ecacy through a
combination with steroids. To the best of our knowledge, this
study is the rst to investigate whether herbal medicines can
be use in a combined treatment with Western medicines for
AD.
Abbreviations
AD: Atopic dermatitis
H&E: Hematoxylin and eosin
HC: Hydrocortisone
TF: Tribuli Fructus
TEWL: Transepidermal water loss
TRPV: Transient receptor potential vanilloid subtype .
Ethical Approval
isstudywasapprovedbytheInstitutionalAnimalCareand
Use Committee at Dongguk University (IACUC--).
Conflicts of Interest
e authors declare no conicts of interest regarding the
publication of this paper.
Authors’ Contributions
Seok Yong Kang, Joo Hyun Nam, Hyo Won Jung, Jong-
Seong Kang, Hyo Sang Bae, and Yong-Ki Park conceived
anddesignedthestudy.SeokYongKangandHyoWonJung
performed the in vivo and in vitro experiments. Joo Hyun
Nam performed the ion channel studies. Jong-Seong Kang,
Young-Ho Kim, Cheong-Weon Cho, and Chong Woon Cho
performed the HPLC analyses. Seok Yong Kang, Joo Hyun
Nam, and Hyo Sang Bae wrote the manuscript. Hyo Sang
Bae and Yong-Ki Park revised it. All the authors read and
approved the nal manuscript.
Acknowledgments
e authors thank everyone who contributed to the study.
is work was supported by the Convergence of Conven-
tional Medicine and Traditional Korean Medicine R&D
Program funded by the Ministry of Health and Welfare
through the Korean Health Industry Development Institute
(SHIDI) (to Hyo Sang Bae, HIC).
References
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