Keywords
Coix lacryma-jobi L. var.
ma-yuen Stapf - Gramineae - Coix seed - Sodium Dodecyl Sulfate - Skin inflammation - Skin dryness
CS Coix seed
PGE2 prostaglandin E2
COX-2 cyclooxygenase 2
ICD irritant contact dermatitis
SDS sodium dodecyl sulfate
GluCer glucosylceramide
H&E hematoxylin, and eosin
HRP horseradish peroxidase
Iba-1 ionized calcium-binding adaptor protein-1
IL-1α interleukin-1α
CMC-Na sodium carboxymethyl cellulose
MRM multiple reaction monitoring
SIM selected ion monitoring
Introduction
Coix seed (CS, seeds of Coix lacryma-jobi L. var. ma-yuen Stapf,
Gramineae) is a traditional Chinese medicine used for invigorating spleen
function, alleviating arthritis, arresting diarrhea, and treating diabetes [1]. CS contains fatty acids [2], saccharides [3], phenolics [4], and lactams [5].
Pharmacological research has demonstrated a wide spectrum of biological activities
of CS, including anti-inflammatory [6]
[7]
[8],
anti-allergic [9], antioxidant [10]
[11]
[12], and antitumor activities
[1]. Several studies using animal models
have demonstrated that CS exerts anti-inflammatory and anti-allergic effects by
regulating pro-inflammatory cytokine expression and suppressing cyclooxygenase 2
(COX-2) and prostaglandin E2 (PGE2) production [13]
[14].
CS is widely used as a therapeutic agent for inflammatory skin disorders, such as
atopic dermatitis [15]
[16] and acute radiation dermatitis [17]. Surfactant-induced irritant contact
dermatitis (ICD) is a common skin disorder. However, to the best of our knowledge,
the efficacy and mechanism of action of CS against surfactant-induced skin disorder
have not been reported to date.
Sodium dodecyl sulfate (SDS), a representative anionic surfactant, is widely used in
domestic cleaning, cosmetic, and pharmaceutical products [18]. Prolonged exposure to SDS induces skin
disorders characterized by skin dryness, scaling, erythema formation, epidermal
hyperplasia, and inflammatory cell infiltration [19]
[20]
[21]. Although the exact mechanism underlying
SDS-induced skin damage is not fully understood, removal of intercellular lipids or
alteration of lipid profiles in the stratum corneum [22]
[23] and the direct toxicity of
SDS on epidermal cells due to the release of inflammatory mediators, such as
interleukin-1α (IL-1α) [24]
[25] and PGE2 [26]
[27]
[28], are recognized as the
major causes of SDS-induced ICD progression. This study aimed to investigate the
preventive effects of CS on skin inflammation and dryness induced by multiple
topical applications of SDS on the dorsal skin of hairless mice and elucidate the
possible mechanism involved.
Results
Eight commercially available compounds (Listed in [Table 1]), previously reported as the bioactive components in CS extract
or its related parts [7]
[8]
[29]
[30]
[31], were selected to quantify their contents
in the CS extract via LC MS/MS. The calibration curves for each standard
were found to be linear with correlation coefficients (R
2
)
greater than 0.999 for the indicated concentration range (Table 3S, Supporting
Information). This quantification was performed in triplicate, and the results are
shown in [Table 1] and [Fig. 1S] (Supporting Information). Among the
evaluated compounds, the content of p-coumaric acid was the highest, followed
by coixol, a characteristic CS alkaloid. Naringenin and luteolin were not detected
in the CS extract. We further analyzed the saccharide and fatty acid contents in the
primary components of CS extract using HPLC and GC-MS, respectively. Three
saccharides (glucose, fructose, and sucrose) and four fatty acids (palmitic acid,
stearic acid, oleic acid, and linoleic acid) were detected, as shown in [Fig. 2S] (Supporting Information). The
presence of these components was validated by comparison with authentic samples.
Table 1 Contents of the test compounds in Coix seed
extract
|
Compounds
|
Contents (μg/g)*
|
RSD (%)
|
|
Protocatechuic acid
|
3.61
|
0.8
|
|
4-Hydroxybenzaldehyde
|
3.24
|
0.8
|
|
Caffeic acid
|
5.78
|
1.6
|
|
p-Coumaric acid
|
29.85
|
0.9
|
|
5,7-Dihydroxychromone
|
4.49
|
5.8
|
|
Naringenin
|
ND
|
ND
|
|
Luteolin
|
ND
|
ND
|
|
Coixol
|
9.10
|
4.9
|
*The content of each compound is presented as
μg/g of CS extract.; RSD: Relative standard deviation
(n=3); ND: not detected, when the signal-to-noise ratio
(S/N) was below 3.
In our preliminary study, we found that daily exposure of mouse dorsal skin to SDS
caused a gradual decrease in epidermal water content, an indicator of skin dryness,
and an increase in erythema index, an indicator of skin inflammation. During the
5-day treatment period, both skin parameters were significantly altered after 5 days
of SDS exposure ([Fig. 3S], Supporting
Information); therefore, this time point was selected for evaluating CS extract
efficacy in this study. The effects of CS against SDS-induced skin dryness and
inflammation were evaluated by measuring the epidermal water content and skin
erythema index. Both skin parameters were measured before the start of SDS treatment
and on the 5th day of the treatment, and the ΔE values are shown
in [Fig. 1]. CS significantly prevented the
SDS-induced reduction in epidermal water content in a dose-dependent manner compared
to that in the SDS-treated group (vehicle group) ([Fig. 1a]). Glucosylceramide (GluCer), a ceramide precursor, was used as
the positive control and showed an effect similar to that of CS ([Fig. 1a]). In addition, oral administration of
500 mg/kg CS significantly prevented the increased erythema index
caused by SDS exposure ([Fig. 1b]). The
change in the erythema index was not significantly different among the low CS
(150 mg/kg), GluCer, and vehicle groups ([Fig. 1b]). By day 5, SDS exposure caused an
increase in the severity of skin scaling ([Fig.
2a]), and the score was significantly increased compared to that of
untreated skin (control group) ([Fig. 2b]).
Administration of 500 mg/kg CS significantly alleviated the severity
of skin scaling compared with that in the vehicle group ([Fig. 2b]). A similar effect was observed in
the GluCer-administered group ([Fig. 2]).
Fig. 1 Effect of CS on SDS-induced reduction of epidermal water
content and elevation of skin erythema index. Epidermal water content and
erythema index of the dorsal skin were measured in each mouse before the
topical application of SDS on day 5. The data are expressed as Δ
epidermal water content (a) and Δ erythema index (b),
calculated by subtracting the values obtained before SDS exposure from the
values obtained on day 5 after SDS exposure. Bars represent
means±standard deviations (n=5 per group);
###
p<0.001 vs. control group;
*
p<0.05,
**
p<0.01,
***
p<0.001
vs. vehicle group; Tukey’s test.
Fig. 2 Effect of CS on SDS-induced skin scaling. Representative images
of the dorsal skin (a) in each group. Photographs were acquired using
a digital camera on day 5 after the start of the SDS topical application.
Arrows, skin scaling; Scale bars, 10 mm. To evaluate the severity of
the SDS-induced skin scaling, the scaling score (b) was derived using
a macroscopic scoring system ranging from 0 to 4 (score 0, none; score 1,
slight; score 2, moderate; score 3, severe; score 4, very severe) on day 5
after the start of SDS topical application. Data points indicate the mode
score for each mouse from five investigators (n=5).
#
p<0.05 vs. vehicle group; post-hoc Steel
test.
To evaluate the effect of CS on epidermal hyperplasia following SDS exposure, we
stained skin tissue sections with hematoxylin and eosin (H&E) and measured
epidermal thickness. By day 5, SDS exposure caused approximately 2-fold higher
epidermal thickening in the vehicle group than in the control group ([Fig. 3a and b]). CS significantly attenuated
the increased epidermal thickness in a dose-dependent manner ([Fig. 3b]). Infiltration of macrophages and
neutrophils is reportedly involved in SDS-induced skin inflammatory reactions [21]. To evaluate the effect of CS extract on
SDS-induced leukocyte infiltration in mouse skin, immunohistochemical staining of
the macrophage marker ionized calcium-binding adaptor protein-1 (Iba-1) and
neutrophil marker Ly-6G/Ly-6C was performed. By day 5, SDS exposure resulted
in macrophage infiltration into the epidermis ([Fig. 3a], Iba–1). The number of Iba-1-positive cells was
significantly higher in the SDS-treated group than in the control group. CS
significantly reduced the number of Iba-1-positive cells in a dose-dependent manner
([Fig. 3c]). However, no significant
difference was observed in the number of neutrophils among the groups (data not
shown).
Fig. 3 Effect of CS on SDS-induced epidermal hyperplasia and
macrophage infiltration in mouse skin. Representative H&E and Iba-1
immunohistochemically stained dorsal skin sections (a) on day 5 after
the start of SDS topical application. Arrow, Iba-1-positive macrophages;
Scale bars, 100 µm. H&E and Iba-1 stained skin
sections were used to quantify the epidermal thickness (b) and the
density of Iba-1-positive macrophages in the skin (c), respectively.
Both quantifications were performed as described in the Materials and
Methods section. Data are expressed as means±standard deviations
(n=5 per group). ##
p<0.01,
###
p<0.001 vs. control group;
*
p<0.05,
**
p<0.01,
***
p<0.001 vs. vehicle
group; Tukey’s test.
Irritant-induced skin inflammatory reactions involve increased production of
inflammatory mediators [32]
[33]. To elucidate how CS attenuates the
SDS-induced skin inflammation, we examined the expression of IL-1α and PGE2,
previously reported to be associated with the SDS-induced inflammatory responses
[24]
[26]. The concentrations of IL-1α and PGE2 in the skin tissue in
the vehicle group were significantly higher than those in the control group. The
increased levels of IL-1α and PGE2 were significantly attenuated by the
administration of 500 mg/kg CS ([Fig. 4a, b]). COX-2 regulates the synthesis of PGE2 from arachidonic
acid. Therefore, we investigated the effect of CS on COX-2 expression using western
blotting. SDS exposure significantly increased the expression of COX-2, and
administration of 500 mg/kg CS markedly attenuated this upregulation
([Fig. 4c]).
Fig. 4 Effect of CS on SDS-induced IL-1α, PGE2, and COX-2
levels in mouse dorsal skin. Dorsal skin was collected on day 5 after the
start of SDS topical application. The production of IL-1α (a)
and PGE2 (b) in the dorsal skin was quantified using ELISA. The COX-2
protein levels (c) in the dorsal skin were analyzed by western
blotting; β-actin was used as the internal standard; Representative
immunoblots are shown above the plot in panel C. Data are expressed as
means±standard deviations (n=5 per group).
#
p<0.05 vs. control group;
*
p<0.05,
**
p<0.01 vs. vehicle group;
Tukey’s test.
Discussion
In this study, we investigated the effect of CS on skin inflammation and dryness
using the SDS-induced ICD model of HR-1 hairless mice and found that CS attenuated
SDS-induced skin dryness, scaling, erythema, epidermal hyperplasia, and inflammatory
cell infiltration. Furthermore, CS inhibited the SDS-induced production of
pro-inflammatory mediators. Our study showed that oral administration of CS can
protect against surfactant-induced inflammation-mediated skin dryness.
Enhanced production of pro-inflammatory mediators in the skin is a major
characteristic of ICD [32]
[33]. PGE2 is one of the most important
mediators implicated in ICD [26]
[27]
[28].
It accelerates the blood flow and enhances vascular permeability, leading to
erythema formation and inflammatory cell infiltration at the site of inflammation
[34]. Additionally, PGE2 increases
keratinocyte proliferation and inhibits keratinocyte differentiation in a
fibroblast-keratinocyte co-culture system [35]. In this study, CS significantly prevented SDS-induced PGE2 production
([Fig. 4b]). During PGE2 synthesis, COX-2
catalyzes the conversion of membrane-released arachidonic acid to PGE2. Our results
also indicated that SDS-induced COX-2 expression is significantly attenuated by CS
administration ([Fig. 4c]). Therefore,
suppressing PGE2 production by inhibiting the COX-2 expression may partially
ameliorate SDS-induced erythema formation, epidermal hyperplasia, and macrophage
infiltration by CS ([Fig. 1b] and [3]). Previous studies have indicated that CS
and its components, such as coixol, caffeic acid, and p-coumaric acid exert
anti-inflammatory effects by suppressing COX-2 [7]
[13]
[36]
[37].
These components were predominantly found in the CS extract ([Fig. 1S] and [Table 1]). However, identification of the compounds responsible for the
protective activity of CS against SDS-induced COX-2-mediated PGE2 production
requires further investigation. IL-1α, the most abundant cytokine present in
the skin, is another key mediator involved in the initiation and maintenance of ICD
[38]. Previous studies have shown that SDS
induces the overexpression of IL-1α both in vitro and in vivo
[[24]
[25]. In response to irritants, IL-1α could be released from
keratinocytes as an initial step in the inflammatory cascade, subsequently
stimulating the production and release of more IL-1α and other
pro-inflammatory cytokines/chemokines from surrounding cells, leading to ICD
development [38]. Hence, inhibition of
IL-1α production may prevent the onset of contact dermatitis. Our results
demonstrated that CS significantly prevented the SDS-induced increase in
IL-1α production ([Fig. 4a]), which
prevents the onset of SDS-induced skin inflammation.
Dried skin with low moisture content is a major hallmark of SDS-induced skin
disorders [19]
[22]. CS administration markedly suppressed the SDS-induced reduction in
epidermal water content ([Fig. 1a]). In
healthy skin, the proliferation and differentiation of keratinocytes are tightly
regulated in the epidermis. However, SDS causes uncontrolled proliferation of
epidermal keratinocytes, leading to the disruption of keratinocyte differentiation,
resulting in the presence of nucleated keratinocytes in the stratum corneum and
scaling on the skin surface [39]. These
incompletely differentiated corneocytes do not have a water-retaining capacity and
are involved in SDS-induced skin dryness. The oral intake of CS reduces the number
of nucleated corneocytes in healthy human skin [40]. This finding and our results ([Fig.
2]
[3b]) suggest CS may alleviate
SDS-induced skin dryness by normalizing the proliferation and differentiation of
epidermal keratinocytes.
Removal of intercellular lipids or alteration of lipid profiles in the stratum
corneum by SDS impairs skin barrier function, which is considered the major cause of
SDS-induced skin dryness [22]
[23]. Ceramides, the major component of
intercellular lipids, play a pivotal role in skin barrier integrity and
moisture-retaining capacity [41]. A previous
study has demonstrated an inverse relationship between ceramide content and skin
dryness after SDS exposure [42]. In this
study, an effect similar to that of CS was observed in the GluCer group ([Fig. 1a]). Linoleic acid, which is abundant in
the CS extract ([Fig. 2S], b), is a
ceramide precursor [43], and elevated ceramide
content in the stratum corneum has been reported in healthy dogs following oral
administration of linoleic acid [44].
Therefore, CS-derived linoleic acid may partly contribute to the preventive effects
of CS against SDS-induced skin dryness by increasing the water-retaining capacity of
the skin.
In conclusion, oral administration of CS mitigates a series of SDS-induced skin
disorders, including skin dryness, scaling, erythema, epidermal hyperplasia, and
macrophage infiltration. The protective effect of CS may be exerted via inhibition
of the production of IL-1α and COX-2-mediated PGE2.
Materials and Methods
Plant materials and extract preparation
The CS plants (Lot# H160722410) were purchased from Koshiro Company Ltd., Osaka,
Japan, and marker compounds were identified according to the Japanese
Pharmacopoeia and industry standards of Kracie Pharmaceutical Ltd. A voucher
specimen (No. 24211) was deposited in the herbarium of Kampo Research
Laboratories, Kracie Pharmaceutical, Ltd. The CS water extract was prepared
according to the Guidance published by the Japanese Pharmaceuticals and Medical
Devices Agency [45]. Briefly, 30 g
of CS, the daily dosage recommended for adults, was soaked in 600 mL
distilled water (20 times the amount of CS, v/w) for 1 h in a
Santo earthenware teapot and concentrated to a final volume of 390 mL
(13 times the amount of CS, v/w) by boiling. The mixture was filtered
through gauze and lyophilized to obtain 840 mg of the extract powder
(yield 2.8%). This extraction was repeated to obtain sufficient extract;
the extracts were homogenized and stored at –20 °C until further
use.
Preparation of the standard and sample solutions
The standards, coixol and 5,7-dihydroxychromone, were obtained from
MedChemExpress Co., Ltd., and ChemFaces Biochemical Co., Ltd., respectively.
Protocatechuic acid, caffeic acid, p-coumaric acid, and
4-hydroxybenzaldehyde were purchased from Sigma-Aldrich Co. LLC. Naringenin and
luteolin were obtained from FUJIFILM Wako Pure Chemical Corporation. All the
standards were of analytical grade, with purity above 98%. Each standard
was accurately weighed and dissolved in HPLC-grade methanol (Wako) to prepare
the respective stock solutions. Calibration standard solutions were prepared by
appropriate dilutions of the mixed stock solution. The sample solution was
prepared as described previously [46].
Briefly, the CS extract (1.0 g) was weighed accurately and dissolved in
10 mL of ultrapure water (Wako). Next, 10 mL of HPLC-grade
acetonitrile (Wako) was added, and the mixture was agitated using a shaker
(Taitec Corporation) for 10 min. A premixed sachet of the QuEChERS
Extraction salts (Agilent Technologies) was further added to the solution and
shaken for 10 min. After centrifugation (3000×g,
10 min), 1 mL of the upper layer solution was transferred into a
10 mL volumetric flask and diluted with water to obtain the sample
solution.
Chemical analysis of the CS extract
The standard and sample solutions were analyzed via LC MS/MS on the
Shimadzu UFLC HPLC system coupled to an LCMS-8030 triple quadrupole mass
spectrometer. The MS/MS analyses of the standards, except for coixol,
were performed in the multiple reaction monitoring (MRM) mode; for coixol,
analysis was in the selected ion monitoring (SIM) mode because no ion pairs were
detected in the MRM mode. The LC MS/MS conditions and the parameters for
each analyte are described in the Supporting Information. The saccharides and
fatty acids in the CS extract were analyzed by HPLC and GC-MS, respectively; the
details are described in the Supporting Information.
Animals
Seven-week-old male hairless HR-1 mice were purchased from Japan SLC Inc., housed
at 24±2°C under a 12/12-h light/dark cycle, and
provided laboratory pellet chow (CE-2, CLEA Japan Inc.) and water ad
libitum. The animal experiments in this study were approved (approval
#190023, August 30, 2019) by the Experimental Animal Care Committee of Kracie
Pharmaceutical, Ltd. and were performed in accordance with the principles of the
Basel Declaration and recommendations in the Guidelines for Proper Conduct of
Animal Experiments.
Treatment
The mice were divided into five groups (n=5) according to their body
weight. Over 4 weeks, the mice in the first and second groups were orally
administered 150 and 500 mg/kg per day of CS extract,
respectively. The dose conversion between animals and humans was performed using
a previously described calculation [47].
The third group received pure GluCer (1 mg/kg per day)
(purity≥99%, Nagara Science) orally and served as a positive
control [25]. Both CS extract and GluCer
were dispersed in 1% (w/v) sodium carboxymethyl cellulose
(CMC-Na) (Wako). The remaining two groups were administered 1% CMC-Na
alone. Topical application of SDS on the dorsal skin was performed using a
previously described method [25]. Briefly,
after 3 weeks of oral administration, medical absorbent cotton (~ca.
3×4 cm, Kawamoto Corporation) containing 3 mL of
10% (w/v) SDS (Wako) was placed in contact with the dorsal mouse
skin for 10 min under isoflurane anesthesia (Wako). SDS exposure was
performed once daily for 5 consecutive days. CS and GluCer were continuously
administered 1 h before SDS exposure. As a control for intact skin, one
group administered 1% CMC-Na alone was not exposed to SDS.
Measurement of skin parameters
Before SDS exposure and on day 5, the skin parameters epidermal water content and
erythema index were measured using Corneometer® CM825 and
Mexameter® MX18 (Courage+Khazaka Electronic),
respectively. All measurements were performed at three sites (upper, middle, and
lower) along the central line of each SDS-treated skin area under isoflurane
anesthesia and were repeated five times on the same position. Changes in each
parameter were calculated using the following formula: index change
(ΔE)=(value after SDS exposure) – (value before SDS
exposure).
Evaluation of the severity of skin scaling
The dorsal mouse skin was photographed on day 5 using a digital camera. To
evaluate the severity of SDS-induced skin scaling using the digital images, a
macroscopic scoring system ranging from 0 to 4 was developed and evaluated
blindly by five investigators as follows: score 0, none (absence of scaling on
the SDS-treated skin area); score 1, slight (appearance of scaling
on<25% of the area); score 2, moderate (appearance of scaling on
25–50% of the area); score 3, severe (appearance of scaling on
50–75% of the area); score 4, very severe (appearance of scaling
on>75% of the area).
Histological and Immunohistochemical analysis
For histologic analysis, the dorsal skin was fixed in Bouin’s fluid
(Wako), embedded in paraffin, sectioned (5 μm thickness), and stained
with H&E. For immunohistochemical analysis, tissues sections (5
μm thick) were stained with anti-Iba-1 or anti-Ly-6G/Ly-6C
antibody (Abcam) using the procedure described in our previous study [48]. Images of the stained skin sections
were captured using a Zeiss Axio Observer Z1 microscope. Epidermal thickness was
calculated by dividing the area of the epidermis by the length of the basal
layer, and the densities of Iba-1- or Ly-6G/Ly-6C-positive cells were
calculated by dividing the number of cells counted by the volume of the counted
area in five randomly sites of each section using the ZEN2.3 software
(Zeiss).
ELISA measurements and Western blotting analysis
The levels of IL-1α (R&D Systems) and PGE2 (ENZO) in the skin
were quantified using ELISA kits according to the manufacturer’s
protocol. The expression levels of COX-2 protein in the skin were measured by
western blotting according to the procedure described previously [48]. Immunoreactive bands were visualized
using an Amersham Imager 680, and band intensities were quantified using
ImageQuant TL8.2 (GE Healthcare).
Statistical analysis
All statistical analyses were performed using EZR (Version 3.5.2) [49]. Statistical comparisons among multiple
groups were performed using one-way analysis of variance followed by
Tukey’s or Kruskal-Wallis test followed by Steel’s post hoc
tests. Differences were considered significant at p<0.05.
Supporting Information
The LC MS/MS conditions and the parameters for each analyte, the procedures
for HPLC analysis of saccharides and GC-MS analysis of fatty acids in the CS
extract, and the time-dependent changes in epidermal water content and erythema
index in both SDS-untreated and treated mouse dorsal skin, are available in the
Supporting Information.
