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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


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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.
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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; and Hyo Sang Bae;
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,
as  antagonists, cytokine antagonists, phosphodiesterase
Evidence-Based Complementary and Alternative Medicine
Volume 2017, Article ID 8312946, 12 pages
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
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 ± 2Cand55 ± 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
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
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
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(in acetone + olive oil) (in acetone + olive oil)
TF or HC treatment
5% Ox 50 ,
12 8 18 24
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 Cinahumidied%CO
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 Turbofecttransfection 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
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
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prepared and contained  mM NaCl,  mM KCl,  mM
HEPES,  mM BaCl2,mMMgCl
2,  mM EGTA, and  mM
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
activate the Orai- current,  𝜇M inositol ,,,-triphosphate
(IP3), which can deplete endoplasmic reticulum (ER)
Ca2+ stores, was added to the pipette solution before the
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
(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,
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)).
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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
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
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
Nor AD 1% TF TF + 0.1% HC 1% HC
Symptom scores
Nor AD 1% TF TF + 0.1% HC 1% HC
30 A∗∗∗
TEWL (g/2B)
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
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
TF extract application to the skin of AD mice can improve
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
AD 1% TF 1% TF + 0.1% HC 1% HCNor
Nor AD 1% TF TF + 0.1% HC 1% HC
e numbers of eosinophils (no.)
Nor AD 1% TF TF + 0.1% HC 1% HC
Mast cells counts (no.)
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
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
AD 1% TF 1% TF + 0.1% HC 1% HCNor
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.
100 50 0 50
Time (sec)
(A) (B) (C)
Normalized current
TF extract 1G/
0.66 ± 0.09
0 200 400 600 800 1000
TF extract 1GA/ G, BTP210
At −120 G6
100 50 0 50 100
0 200 400 600
Time (sec)
Normalized current
TF extract 1G/
TF extract 1G/
0.37 ± 0.12
R.R 50 -
2-APB 100 - At −100 G6
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)aerTFextracttreatment()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
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)
−−−−− −
NAG activity (mU/mL)
TF0.2 TF0.5 2-APB
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
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0
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: Atopic dermatitis
H&E: Hematoxylin and eosin
HC: Hydrocortisone
TF: Tribuli Fructus
TEWL: Transepidermal water loss
TRPV: Transient receptor potential vanilloid subtype .
Ethical Approval
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
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.
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).
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... TT is believed to be a scavenger of free radicals due to its active constituents (saponins, alkaloids, flavonoids), which might improve sperm parameters, particularly under stress conditions [57]. The effect of TT on the Ca 2+ channel could be a possible reason for its positive impact on sperm motility [58]. TT may improve semen parameters because of its strong antioxidant 16 Andrologia activity by regulating Nuclear factor erythroid 2-related factor (Nrf-2) and Heme-oxygenase-1 (HO-1) signalling. ...
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Tribulus terrestris L. (TT) is a traditional medicinal plant, which belongs to the Zygophyllaceae family. TT extracts have been widely used for diuretic, analeptic, aphrodisiac, and profertility properties. To quantitatively evaluate the profertility and aphrodisiac effects of Tribulus terrestris L., we undertook the present meta-analyses on published data. A thorough literature screening was performed to identify articles evaluating the effect of TT on spermatogenesis, male fertility, reproductive, and aphrodisiac parameters. We shortlisted 30 relevant studies conducted on humans and rodents. Meta-analyses were conducted to evaluate the quantitative impact of TT on various fertility parameters. In case of humans, the pooled analysis on 133 subjects showed significant improvements in sperm concentration (SDM = 0:624, 95% CI = 0:13 to 1.117, p = 0:013) and sperm motility (SDM = 0:742, 95% CI = 0:331 to 1.152, p = 0:001). TT resulted in nonsignificant increases in testosterone and LH and a nonsignificant decrease in FSH. Similar to the above, TT improved sperm count, sperm motility, and sperm viability in rodents with normal or compromised fertility. The effect on hormone levels was less credible with frequent variations across studies and animal models. The aphrodisiac activity was studied in castrated animal models or normal rodents, both of which showed significant improvements in mounting frequency and intromission frequency and significant declines in mounting latency and intromission latency. These meta-analyses suggested that TT possesses profertility and aphrodisiac activities.
... It was reported that ORAI channel was involved in MRGPRX2-regulated mast cell activation, and inhibition of it might be the potential treatment method for MRGPRX2mediated disorders [13]. Meanwhile, Tribuli Fructus extract could suppress skin inflammation in atopic dermatitis mice through regulating calcium channels and mast cell activation by targeting ORAI-1 [13,33]. In this study, we found that the protein expression levels of MRGPRX2, ORAI-1, and ORAI-2 in the skin tissues were greatly suppressed by Celastrol, but the decrease of MRGPRX2/ORAI was promoted by MRGPRX2 OE . ...
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Background: Atopic dermatitis is a common dermatological disease, and mast cell degranulation is believed to be related with the progression of atopic dermatitis. Mas-related G protein-coupled receptor-X2 (MRGPRX2), and calcium release-activated calcium channel protein 1-2 (ORAI-1, ORAI-2) are involved in mast cell degranulation. Celastrol is an active monomer of Tripterygium wilfordii, and it presents an antiatopic role. Methods: 2,4-Dinitrofluorobenzene (DNFB) and compound 48/80 (C 48/80) were used to establish a slow and acute scratching animal model, respectively. Hematoxylin-eosin and toluidine blue staining was used to investigate tissue injury. Inflammatory factor concentration was measured with ELISA. The expression of MRGPRX2, ORAI-1, and ORAI-2 was detected with immunohistochemistry (IHC) staining. Gene expression profiling and microRNA array were performed to investigate gene differential expression. Results: Celastrol greatly inhibited atopic dermatitis-related tissues injury, mast cell production, histamine release, scratching level, inflammatory factor expression, and activation of MRGPRX2/ORAI axis in the DNFB-induced atopic dermatitis model. The influence of Celastrol on atopic dermatitis was remarkably reversed by overexpression of MRGPRX2. Conclusion: We found that the improvements of atopic dermatitis caused by Celastrol were reversed by treatment with MRGPRX2OE, indicating that Celastrol might affect atopic dermatitis through MRGPRX2. This study might provide a novel thought for the prevention and treatment of atopic dermatitis by regulating MRGPRX2.
... Skin symptoms were recorded on day 15. The severities of skin lesions were evaluated macroscopically as 4 levels: 0, no symptoms; 1, mild; 2, moderate; and 3, severe using the previously described scoring standards of atopic/ eczema dermatitis syndrome concerning dryness/scaling, erythema/ hemorrhage, and excoriation/erosion (Kang et al., 2017). ...
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Atopic dermatitis (AD), characterized by severe pruritus, immune imbalance, and skin barrier dysfunction, has a high incidence worldwide. Recent evidence has shown that the modulation of gut microbiota is crucial for alleviating clinical symptoms of AD. Tremella fuciformis polysaccharides (TFPS) have been demonstrated to have a variety of biological activities such as immunomodulatory, anti-tumor, antioxidant, anti-inflammatory, neuroprotective, hypoglycemic and hypolipidemic effects. However, their effects on AD treatment have never been investigated. In this study, we compared the therapeutic effects of topical or oral administration of TFPS on AD in dinitrofluorobenzene (DNFB)-induced AD mice. Both topical application and oral administration of TFPS led to improvement on transdermal water loss, epidermal thickening, and ear edema in AD mice, but the oral administration showed significantly better efficacy than the topical application. The TFPS treatment increased the proportion of CD4 (+) CD25 (+) Foxp3 (+) regulatory T cells in mesenteric lymph nodes. Additionally, the non-targeted metabolomics and sequencing of 16S rDNA amplicons were performed, revealing metabolite modulation in feces and changed composition of gut microbiota in mice, which were induced for AD-like disorder and treated by oral administration of TFPS. Collectively, these data suggest that the oral administration of TFPS may constitute a novel effective therapy for AD, with underlying mechanisms associated with the regulation of immune response, and improvement of both metabolism and the composition of intestinal microbiota.
... Traditional medicinal claims about T. terrestris are well supported by the pharmacological studies wherein it is widely applied to treat inflammation [13], drying of skin and itchiness [15], heart and blood vessel related ailments [16], carcinomas [17], infections induced by microbes [18], oxidative damage [19], hormonal problems [20], and help to repair muscles [21]. The reported activities were related to saponins, flavonoids, and alkaloids [21,22]. ...
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Tribulus terrestris L. belongs to the family Zygophyllaceae and integral part of various ancient medicinal systems including Chinese, Indian, and European to combat various health ailments. The aim of the present study was to assess the phytochemical constituents, in vitro antioxidant activity using DPPH, FRAP, and H2O2 assays, in vitro anticancer activity using MTT assay, and in vitro and in vivo anti-inflammatory properties of T. terrestris. The acute and sub-acute toxicity of extracts exhibiting most biological potential was examined using murine models. Liquid–liquid partitioning followed by RP–HPLC sub-fraction of crude extract was performed. After that, ESI-MS/MS analysis was done for the timid identification of bioactive metabolites responsible for bioactivities of sub-fractions and HPLC analysis to quantify the compounds using external standards. Among all extracts, T. terrestris methanol extract was noted to hold maximum phenolic (341.3 mg GAE/g) and flavonoid (209 mg QE/g) contents, antioxidant activity in DPPH (IC50 71.4 µg/mL), FRAP (35.3 mmol/g), and H2O2 (65.3% inhibition) assays, anti-inflammatory activities in vitro at 400 µg/mL (heat-induced hemolysis, % inhibition 68.5; egg albumin denaturation, % inhibition 75.6%; serum albumin denaturation, % inhibition 80.2), and in vivo at 200 mg/kg (carrageenan-induced paw edema, % inhibition 69.3%; formaldehyde-induced paw edema, % inhibition 71.3%) and anticancer activity against breast cancer cell (MCF-7) proliferation (IC50 74.1 µg/mL). Acute and sub-acute toxicity studies recorded with no change in body weight, behavior, hematological, serum, and histopathological parameters in treated rats with T. terrestris methanol extracts when compared to control group. Fraction B obtained through liquid–liquid partitioning resulted in more bioactive potential as compared to the parent methanol extract. RP–HPLC analysis of fraction B resulted with four sub-fractions (TBTMF1-TBTMF4), wherein TBTMF3 delineated notable bioactive capabilities as compared to other fractions and parent methanol extract. ESI-MS/MS analysis of TBTMF3 resulted with tentative identification of myricetin, rutin, liquitrigenin, physcion, and protodioscin. It can be stated that T. terrestris is a potential bearing herb and findings of current study further verify the claims made in ancient medicinal systems. However, after investigation of each identified compound, it must be considered for drug discovery.
... 14 Of significant translational relevance, TRPV3 expression was found to be increased in lesional skin of atopic dermatitis (AD) patients as well as in pruritic post-burn lesions on the mRNA level. 13,15 Inhibition of TRPV3 by various compounds has been proven to be effective in ameliorating AD-like symptoms in animal models, [16][17][18][19][20][21] and the channel has also been linked to proteinaseactivated receptor 2. 22,23 A more recent report has expanded on these results, by showing that TRPV3 expression is also increased on the protein level in AD lesions, and that heat-induced itch in AD patients is dependent on TRPV3. Further, keratinocytes isolated from lesional skin of AD patients showed increased sensitivity to heat in a TRPV3-dependent manner, which resulted in release of pruritogens. ...
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TRPV3 (transient receptor potential vanilloid 3) is a pro-inflammatory ion channel mostly expressed by keratinocytes of the human skin. Previous studies have shown that the expression of TRPV3 is markedly upregulated in the lesional epidermis of atopic dermatitis (AD) patients suggesting a potential pathogenetic role of the ion channel in the disease. In the current study, we aimed at defining the molecular and functional expression of TRPV3 in non-lesional skin of AD patients as previous studies implicated that healthy-appearing skin in AD are markedly distinct from normal skin with respect to terminal differentiation and certain immune function abnormalities. By using multiple, complementary immunolabeling and RT-qPCR technologies on full-thickness and epidermal shave biopsy samples from AD patients (lesional, non-lesional) and healthy volunteers, we provide the first evidence that the expression of TRPV3 is markedly upregulated in non-lesional human AD epidermis, similar to lesional AD samples. Of further importance, by using the patch-clamp method on cultured healthy and non-lesional AD keratinocytes, we also show that this upregulation is functional as determined by the significantly augmented TRPV3-specific ion current (induced by agonists) on cultured non-lesional AD keratinocytes when compared to healthy ones.
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Itching is a sensory phenomenon characterized by an unpleasant sensation that makes you want to scratch the skin, and chronic itching diminishes the quality of life. In recent studies, multiple transient receptor potential (TRP) channels present in keratinocytes or nerve endings have been shown to engage in the propagation of itch signals in chronic dermatological or pruritic conditions, such as atopic dermatitis (AD) and psoriasis (PS). TRPV3, a member of the TRP family, is highly expressed in the epidermal keratinocytes. Normal TRPV3 signaling is essential for maintaining epidermal barrier homeostasis. In recent decades, many studies have suggested that TRPV3 contributes to detecting pruritus signals. Gain-of-function mutations in TRPV3 in mice and humans are characterized by severe itching, hyperkeratosis, and elevated total IgE levels. These studies suggest that TRPV3 is an important channel for skin itching. Preclinical studies have provided evidence to support the development of TRPV3 antagonists for treating inflammatory skin conditions, itchiness, and pain. This review explores the role of TRPV3 in chronic pruritus, collating clinical and experimental evidence. We also discuss underlying cellular and molecular mechanisms and explore the potential of TRPV3 antagonists as therapeutic agents.
The plant Tribulus Terrestris commonly known as Gokhru or Sarata is used in household medicine as a tonic, Aphrodisiac, Palliative, Astringent, Gastric, anti-infective medicine. The literature when surfaced shows use of leaf, stem, shoots, roots, fruit and whole plant extract of the medicinal plant Tribulus Terrestris for various nanoparticles synthesis viz. Silver, gold, nickel, nickel oxide. The synthesised nanoparticles were reported to show various bioactivities viz. antibacterial, antifungal activities. The photocatalytic and cytotoxicity effects of the synthesized nanoparticles were also reported in the recent literatures. This review aims to give a comprehensive overview of various researchers' efforts towards the biosynthesis of various nanoparticles using the extracts of this medicinal plant Tribulus Terrestris during 2012–2021.
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Over the past few decades, complementary and alternative medicine (CAM) using herbs, or their active constituents have garnered substantial attention in the management of a chronic and relapsing inflammatory skin disorder called atopic dermatitis (AD), particularly in attenuating disease recurrence and maintaining long-term remission. In Eastern Asian countries including China, Korea and Taiwan, herbal medicine available in both topical and oral preparation plays a significant role in treating skin diseases like AD as they possibly confer high anti-inflammatory properties and immunomodulatory functions. Conventional murine models of AD have been employed in drug discovery to provide scientific evidence for conclusive and specific pharmacological effects elicited by the use of traditional herbs and their active constituents. Coupled with the goal to develop safe and effective novel therapeutic agents for AD, this systematic review consists of a summary of 103 articles on both orally and topically administered herbs and their active constituents in the murine model, whereby articles were screened and selected via a specialized framework known as PICO (Population, Intervention, Comparator and Outcome). The objectives of this review paper were to identify the efficacy of oral and topical administered herbs along with their active constituents in alleviating AD and the underlying mechanism of actions, as well as the animal models and choice of inducer agents used in these studies. The main outcome on the efficacy of the majority of the herbs and their active constituents illustrated suppression of Th2 response as well as improvements in the severity of AD lesions, suppression of Immunoglobulin E (IgE) concentration and mast cell infiltration. The majority of these studies used BALB/c mice followed by NC/Nga mice (commonly used gender–male; commonly used age group – 6–8 weeks). The most used agent in inducing AD was 2, 4-Dinitrochlorobenzene (DNCB), and the average induction period for both oral and topical administered herbs and their active constituents in AD experiments lasted between 3 and 4 weeks. In light of these findings, this review paper could potentially assist researchers in exploring the potential candidate herbs and their active constituents using murine model for the amelioration of AD.
The Chemistry inside Spices and Herbs: Research and Development brings comprehensive information about the chemistry of spices and herbs with a focus on recent research in this field. The book is an extensive 2-part collection of 20 chapters contributed by experts in phytochemistry with the aim to give the reader deep knowledge about phytochemical constituents in herbal plants and their benefits. The contents include reviews on the biochemistry and biotechnology of spices and herbs, herbal medicines, biologically active compounds and their role in therapeutics among other topics. Chapters which highlight natural drugs and their role in different diseases and special plants of clinical significance are also included. Part II continues from the previous part with chapters on the treatment of skin diseases and oral problems. This part focuses on clinically important herbs such as turmeric, fenugreek, ashwagandha (Indian winter cherry), basil, Terminalia chebula (black myrobalan). In terms of phytochemicals, this part presents chapters that cover resveratrol, piperine and circumin.
The Chemistry inside Spices & Herbs: Research and Development brings comprehensive information about the chemistry of spices and herbs with a focus on recent research in this field. The book is an extensive 2-part collection of 20 chapters contributed by experts in phytochemistry with the aim to give the reader deep knowledge about phytochemical constituents in herbal plants and their benefits. The contents include reviews on the biochemistry and biotechnology of spices and herbs, herbal medicines, biologically active compounds and their role in therapeutics among other topics. Chapters which highlight natural drugs and their role in different diseases and special plants of clinical significance are also included. Part II continues from the previous part with chapters on the treatment of skin diseases and oral problems. This part focuses on clinically important herbs such as turmeric, fenugreek, ashwagandha (Indian winter cherry), basil, Terminalia chebula (black myrobalan). In terms of phytochemicals, this part presents chapters that cover resveratrol, piperine and circumin. Audience: This book is an ideal resource for scholars (in life sciences, phytomedicine and natural product chemistry) and general readers who want to understand the importance of herbs, spices and traditional medicine in pharmaceutical and clinical research.
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The current study was carried out to evaluate the genotoxic aspects of the aqueous extracts of the Tribulus terrestris fruits by comet assay and cytogenetic procedures conditions on cultured human peripheral blood lymphocyte. After the treatment of the lymphocytes with four concentrations of the aqueous fruit extract of T. terrestris (10, 20, 40 and 80 mg/L) for 24 hours it was noticed that, the presence of micronuclei and/or chromosomal aberration were monitored and a significant increase of comet cells at high concentration of T. terrestris extract 80 mg/L. Also, this study showed that the presence of micronuclei, chromosomal aberration as a chromosomal gap, fragmentation, stickiness and necrotic cells were appeared and increased with high concentrations of T. terrestris fruits extract (40-80 mg/L). On the other hand, no significant difference was observed with the low concentration of the extract (10-20 mg/L) as compared with control. The current study refers to the ability of the extract of T. terrestris fruits to do damage in the target DNA at the higher concentrations. Thus, it could be considered that the aqueous extracts of the T. terrestris fruits have genotoxic effect in the therapeutic protocols if it used in high doses.
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Objective: To examine the effects of Tribulus terrestris L. (T. terrestris) extract on the modulation of calcium channels to evaluate its use in topical agents for treatment of atopic dermatitis. Methods: The 70% methanol extract of T. terrestris was prepared. Human HEK293T cells with over-expressed calcium release-activated calcium channel protein 1 (Orai1), transient receptor potential vanilloid 1, or transient receptor potential vanilloid 3 (TRPV3) were treated with T. terrestris extract. Modulation of ion channels was measured using a conventional whole-cell patch-clamp technique. Results: T. terrestris extract (100 μg/mL) significantly inhibited Orai1 activity in Orai1-stromal interaction molecule 1 co-overexpressed HEK293T cells. In addition, T. terrestris extract significantly increased the TRPV3 activity compared with 2-Aminoethyl diphenylborinate (100 μmol/L), which induces the full activation of TRPV3. Conclusions: Our results suggest that T. terrestris extract may have a therapeutic potential for recovery of abnormal skin barrier pathologies in atopic dermatitis through modulating the activities of calcium ion channels, Orai1 and TRPV3. This is the first study to report the modulatory effect of a medicinal plant on the function of ion channels in skin barrier.
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According beneficial effects of Tribulus terrestris (TT) extract on tissue damage, the present study investigated the influence of hydroalcoholic extract of TT plant on cisplatin (CIS) (EBEWE Pharma, Unterach, Austria) induced renal tissue damage in male mice. Thirty mice were divided into five groups (n = 6). The first group (control) was treated with normal saline (0.9% NaCl) and experimental groups with CIS (E1), CIS + 100 mg/kg extract of TT (E2), CIS + 300 mg/kg extract of TT (E3), CIS + 500 mg/kg extract of TT (E4) intraperitoneally. The kidneys were removed after 4 days of injections, and histological evaluations were performed. The data were analyzed using one-way analysis of variance followed by Tukey's post-hoc test, paired-sample t-test, Kruskal-Wallis and Mann-Whitney tests. In the CIS treated group, the whole kidney tissue showed an increased dilatation of Bowman's capsule, medullar congestion, and dilatation of collecting tubules and a decreased in the body weight and kidney weight. These parameters reached to the normal range after administration of fruit extracts of TT for 4 days. The results suggested that the oral administration of TT fruit extract at dose 100, 300 and 500 mg/kg body weight provided protection against the CIS induced toxicity in the mice.
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The oral cavity provides an entrance to the alimentary tract to serve as a protective barrier against harmful environmental stimuli. The oral mucosa is susceptible to injury because of its location; nonetheless, it has faster wound healing than the skin and less scar formation. However, the molecular pathways regulating this wound healing are unclear. Here, we show that transient receptor potential vanilloid 3 (TRPV3), a thermosensitive Ca(2+)-permeable channel, is more highly expressed in murine oral epithelia than in the skin by quantitative RT-PCR. We found that temperatures above 33°C activated TRPV3 and promoted oral epithelial cell proliferation. The proliferation rate in the oral epithelia of TRPV3 knockout (TRPV3KO) mice was less than that of wild-type (WT) mice. We investigated the contribution of TRPV3 to wound healing using a molar tooth extraction model and found that oral wound closure was delayed in TRPV3KO mice compared with that in WT mice. TRPV3 mRNA was up-regulated in wounded tissues, suggesting that TRPV3 may contribute to oral wound repair. We identified TRPV3 as an essential receptor in heat-induced oral epithelia proliferation and wound healing. Our findings suggest that TRPV3 activation could be a potential therapeutic target for wound healing in skin and oral mucosa.-Aijima, R., Wang, B., Takao, T., Mihara, H., Kashio, M., Ohsaki, Y., Zhang, J.-Q., Mizuno, A., Suzuki, M., Yamashita, Y., Masuko, S., Goto, M., Tominaga, M., Kido, M. A. The thermosensitive TRPV3 channel contributes to rapid wound healing in oral epithelia.
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Tribulus terrestris (family Zygophyllaceae), commonly known as Gokshur or Gokharu or puncture vine, has been used for a long time in both the Indian and Chinese systems of medicine for treatment of various kinds of diseases. Its various parts contain a variety of chemical constituents which are medicinally important, such as flavonoids, flavonol glycosides, steroidal saponins, and alkaloids. It has diuretic, aphrodisiac, antiurolithic, immunomodulatory, antidiabetic, absorption enhancing, hypolipidemic, cardiotonic, central nervous system, hepatoprotective, anti-inflammatory, analgesic, antispasmodic, anticancer, antibacterial, anthelmintic, larvicidal, and anticariogenic activities. For the last few decades or so, extensive research work has been done to prove its biological activities and the pharmacology of its extracts. The aim of this review is to create a database for further investigations of the discovered phytochemical and pharmacological properties of this plant to promote research. This will help in confirmation of its traditional use along with its value-added utility, eventually leading to higher revenues from the plant.
Atopic dermatitis (AD), also known as atopic eczema, is a chronic relapsing inflammatory skin condition. Incidence of AD has increased 2- to 3-fold in industrialized nations, impacting approximately 15% to 20% of children and 1% to 3% of adults worldwide. AD has a wide-ranging impact on a patient's quality of life and the burden from direct and indirect costs (approximately $37.7 billion in out-of-pocket costs) is shared by the families and caregivers of patients with AD. This article reviews the epidemiology, burden of disease, pathophysiology, and diagnostic criteria important for early diagnosis and treatment. New insights related to the genetic, immunologic, and environmental impacts of AD have created new treatment opportunities. Nonpharmacologic and pharmacologic interventions are discussed, with an emphasis on emerging treatments for AD. Healthcare providers play an important role in the management of AD to improve economic and clinical outcomes. Treatment strategies need to be individualized with a strong emphasis on patient education and self-management strategies to optimize outcomes and reduce unnecessary costs associated with the management of AD.
Primary vagal afferent neurons express a multitude of thermosensitive ion channels. Within this family of ion channels, the heat-sensitive capsaicin receptor (TRPV1) greatly influences vagal afferent signaling by determining threshold for action-potential initiation at peripheral endings while controlling temperature sensitive forms of glutamate release at central vagal terminals. Genetic deletion of TRPV1 does not completely eliminate these temperature-dependent effects, suggesting involvement of additional thermosensitive ion channels. The warm-sensitive, calcium permeable, ion channel TRPV3 is commonly expressed with TRPV1; however the extent to which TRPV3 is found in vagal afferent neurons is unknown. Here we begin to characterize the genetic and functional expression of TRPV3 in vagal afferent neurons using molecular biology (RT-PCR and RT-qPCR) in whole nodose and isolated neurons and fluorescent calcium imaging on primary cultures of nodose ganglia neurons. We confirmed low level TRPV3 expression in vagal afferent neurons and observed direct activation with putative TRPV3 agonists eugenol, ethyl vanillin (EVA), and farnesyl pyrophosphate (FPP). Agonist activation stimulated neurons also containing TRPV1 and was blocked by ruthenium red. FPP sensitivity overlapped with EVA and eugenol but represented the smallest percentage of vagal afferent neurons; and was the only agonist that did not stimulate neurons from TRPV3 -/- mice, suggesting FPP has the highest selectivity. Further, FPP was predictive of enhanced responses to capsaicin, EVA, and eugenol in rats. From our results we conclude TRPV3 is expressed in a discrete subpopulation of vagal afferent neurons and may contribute to vagal afferent signaling either directly or in combination with TRPV1.
Regular keratinocyte differentiation is crucial for the formation of an intact epidermal barrier and is triggered by extracellular calcium. Disturbances of epidermal barrier formation and aberrant keratinocyte differentiation are involved in the pathophysiology of several skin diseases, such as psoriasis, atopic dermatitis, basal and squamous skin cancer, and genetic skin diseases such as Darier's disease and Olmstedt syndrome. In this review, we summarize current knowledge about the underlying molecular mechanisms of calcium-induced differentiation in keratinocytes. We provide an overview of calcium's genomic and non-genomic mechanisms to induce differentiation and discuss the calcium gradient in the epidermis, giving rise to cornified skin and lipid envelope formation. We focus on the calcium-sensing receptor, transient receptor potential channels, and STIM/Orai as the major constituents of calcium sensing and calcium entry in the keratinocytes. Finally, skin diseases linked to impaired differentiation will be discussed, paying special attention to disturbed TRP channel expression and TRP channel mutations.
In the skin, cannabinoid lipids, whether of endogenous or exogenous origin, are capable of regulating numerous sensory, homeostatic and inflammatory events. Although many of these effects are mediated by metabotropic CB receptors, a growing body of evidence has revealed that multiple members of the transient receptor potential (TRP) ion channel family can act as "ionotropic cannabinoid receptors". Furthermore, many of these same TRP channels are intimately involved in cutaneous processes that include the initiation of pain, temperature, and itch perception, the maintenance of epidermal homeostasis, the regulation of hair follicles and sebaceous glands, and the modulation of dermatitis. Ionotropic cannabinoid receptors therefore represent potentially attractive targets for the therapeutic use of cannabinoids to treat sensory and dermatological diseases. Furthermore, the interactions between neurons and other cell types that are mediated by cutaneous ionotropic cannabinoid receptors are likely to be recapitulated during physiological and pathophysiological processes in the central nervous system and elsewhere, making the skin an ideal setting in which to dissect general complexities of cannabinoid signaling.