A Traditional Medicine-Based Combination of Bamboo Salt and Egg White-Chalcanthite Ameliorates Atopic Dermatitis

*Corresponding Author:

Kyung Seuk Song

Korea Conformity Laboratories, Gaetbeol-ro 145 beon-gil, Yeonsu-gu, Incheon 21999, Korea

E-mail: [email protected]

Abstract

Bamboo salt and egg white-chalcanthite mixture has been prescribed for relieving the inflammation of atopic dermatitis in Hamyang, Gyeongsangnam-do, Korea. This study was conducted to demonstrate the effect of atopic dermatitis on cell and atopic animal models using a combination of bamboo salt with egg white-chalcanthite, which have been used as folk remedies for inflammatory conditions. Based on the in vitro experiment, the 1:20 mixture of egg white-chalcanthite and bamboo salt was shown to inhibit the release of beta-hexosaminidase and histamine in rat basophilic leukemia cell line RBL-2H3 by sensitizing immunoglobulin E and inducing allergic responses by dinitrophenyl as well as the expression of inflammatory cytokines and inflammatory mediators in a dose-dependent manner in lipopolysaccharide-treated mouse macrophage cell line RAW 264.7 cells. Furthermore, egg white-chalcanthite with bamboo salt and its combination at different mixing ratios showed a decrease in inflammatory mediators. Dinitrochlorbenzene was applied to NC/Nga mice for 6 w to induce the atopic in vivo model. After 4 w administration of the test substance, the dermatitis decreased visually and dermal hyperplasia/inflammation, mast cell/eosinophils counts and degree of nerve fiber infiltration were decreased according to histopathological examination. In addition, the secretion rate of interferon gamma in high-dose treated group was lower than that in atopic dermatitis control group. These results show that bamboo salt with egg white-chalcanthite effectively relieves skin inflammation in atopic dermatitis and it might be a promising mineral agent for relieving the symptoms of atopic dermatitis.

Keywords

Atopic dermatitis, bamboo salt, egg white chalcanthite, inflammation

Atopic Dermatitis (AD) is a multifactorial immune
disorder that causes serious symptoms including severe
skin irritation. Skin barrier anomalies and overactive
immune systems secrete modulators to the skin surface,
causing inflammation and skin irritation[1]. The rash on
the skin of patients with AD is visible only near the
dermis. Even though the skin looks normal, there may
still be inflammation near the dermal layer of the skin.
Additionally, scratching the itchy skin will peel off the
stratum corneum of the skin surface, causing bacteria,
viruses and allergens to enter the body followed by
secretion of immunological elements close to the
skin surface causing irritation to the skin and mucous
membranes, accompanied by redness and itching[2].

AD is one of the several chronic recurrent skin
diseases that require continuous treatment. In general,
antihistamines and atopic treatments can alleviate the
tingling sensation in the skin but fail to serve as an
effective long-term remedy[3]. Studies investigating the effect of suppressing atopy and improving atopic
symptoms using natural products and oriental medicine
have been carried out in Korea with a goal to develop
novel and specific drugs[4-7]. Furthermore, it has
been reported that a healthy balance between natural
moisturizing factors and dryness of the skin may be
critical in maintaining skin health[8]. Therefore, studies
testing the efficacy of certain nutrients or dietary
materials containing nutrients related to dryness of
skin have been actively conducted. In the past, health
supplements containing food ingredients with medicinal
value were developed and marketed. However, in
recent times, functional foods or cosmetics made from food extracts are receiving promising medical
attention[9,10]. Despite several studies in this field, no
breakthrough treatment for atopy has yet been devised.

The mixture of bamboo salt and egg white-chalcanthite
has been prescribed for relieving the inflamed
condition in AD in Hamyang-eup, Hamyang-gun and
Gyeongsangnam-do, Korea in past decades. In this
study, we investigated the efficacy of bamboo salt and
egg white-chalcanthite composition, which was used
for prevention and treatment of skin diseases by folk
medicine. After confirming the effect of the substance in vitro, the efficacy of dermatitis treatment was
evaluated to confirm the possibility of development
of the substance as a medication for AD in an animal
model.

Materials and Methods

Preparation of the test substance:

The chalcanthite, natural mineral containing Copper
sulfate pentahydrate (CuSO₄.5H₂O) was heated at 140°
for 4 h to be completely dehydrated, cooled at room temperature and then powdered into a mortar (fig.
1A-fig. 1C). Chalcanthite powder and egg white was
mixed at a ratio of 1:0.7, cooled as shown in fig. 1D and
then powdered again with mortar. The 9^th step molten
bamboo salt was produced by the following procedure;
sun-dried salt, obtained from the western seacoast
of Korea was added into the bamboo (70~100 mm
φ) and capped with red clay; bamboo with sun-dried
salt was piled up one by one into brazier and burned
at 800° with pine wood; remaining salt pillars were
crushed; salt was put into new bamboo slice, sealed
with red clay, burned and crushed 8 times, similar to
that in first step; in the final 9th step, melting, pine resin
was added to the salt and then heated up to 1400°;
liquefied salt sample was cooled at room temperature
for 12 h and finely pulverized as shown in fig. 1E.
The samples, egg white-chalcanthite, bamboo salt and
their combination (fig. 1F), were uniformly dissolved
in sterilized distilled water at a concentration of 100
mg/ml and the materials were filtered with 0.2 μm pore
membrane (Advantec^®, 25CS020AS). The test solution
was prepared immediately prior to treatment.

Fig. 1: Photographic data of the test materials

Standard compound analysis:

Copper was selected as the major component of the
bamboo salt with egg white-chalcanthite because it is a
suitable mineral for quality control. Copper was analysed
by Atomic Absorption Spectroscopy (AAS), AA280FS,
varian, USA system. Analysis was performed using
air/acetylene flame type Copper Hollow Cathode (Cu
HC) lamp (varian, USA) under 13.98 l/min gas flow,
1.78 l/min acetylene, 324.8 nm absorbance wavelength
and 5 mA current. The standard material was used
International Comparison Program (ICP) quality
control standard #3 (AccuStandard, Inc.). Egg whitechalcanthite,
bamboo salt, egg white-chalcanthite and
bamboo salt mixture (1:20) was analysed respectively.
Each test substance was prepared with 1 % (0.3 g/30 ml
distilled water, filtered through a 0.2 μm syringe filter
and then injected with nitric acid to a concentration of
2 % (v/v). The 9^th molten bamboo salt was not diluted
because egg white-chalcanthite and mixture was diluted
100 and 2000 times, respectively, with 2 % nitric acid.

Cell culture:

Rat Basophilic Leukemia (RBL-2H3) cell line and
a mouse macrophage cell line (RAW 264.7), were
purchased from the Korean Cell Line Bank (Seoul,
Korea). Dulbecco’s Modified Eagle’s Medium (DMEM)
containing 10 % heat-inactivated Fetal Bovine Serum
(FBS), 2 mm L-glutamine, 100 units/ml penicillin and
100 μg/ml streptomycin were used to maintain the
cell culture. Both cell lines were incubated under 5 %
Carbon dioxide (CO₂), at 37°.

Cell viability assay:

A preliminary test was conducted by treating the two cell
lines with 200, 40, 8, 1.6 and 0.32 μg/ml concentration
of Cell Counting Kit-8 (CCK-8) (Dojindo Molecular
Technologies, Inc., kamimashiki gun, Kumamoto,
Japan, lot no. KL709). Cells were seeded at 2×10⁴ cells/
well in a 96-well plate and incubated in a CO₂ incubator
at 37° for 24 h. After growth medium was removed,
the cells were treated with the test solution (200, 40, 8,
1.6, 0.32 μg/ml) and cultured for 24 h. CCK-8 solution
(10 μl) was added to the cell culture medium (100 μl).
After incubation for 2 h in a CO₂ incubator, absorbance
was measured at 450 nm using a microplate reader
(Molecular devices, SoftMax Pro5, USA). Based on the
measured optical density value, the cell viability was
calculated and the final Lethal Concentration 50 (LC₅₀) was determined.

Beta (β)-hexosaminidase assay:

Cells suspended in culture medium were plated at 4×10⁵ cells/well in a 6-well plate and incubated
in a 37°, CO₂ incubator for 8 h. After removing
the existing medium, anti-Dinitrophenyl (DNP)
Immunoglobulin E (IgE) diluted to 0.1 μg/ml was
added to the serum-free medium for sensitization.
Test solutions of egg white-chalcanthite, bamboo
salt, 100 mg/ml ketotifen fumarate and mixture
of egg white-chalcanthite and bamboo salt
(1:10, 1:15, 1:20) were treated overnight. After
washing three times with Tyrode buffer (135
mM Sodium chloride (NaCl), 5 mM Potassium
chloride (KCl), 20 mM 4-(2-Hydroxyethyl)-1-
Piperazineethanesulfonic Acid (HEPES), 5.6 mM
glucose, 1.8 mM Calcium chloride (CaCl₂), 1
mM Magnesium chloride (MgCl₂), 0.05 % Bovine
Serum Albumin (BSA), pH 7.4), 0.01 μg/ml DNPBSA
(antigen) was added and incubated for 1 h.
The supernatant was collected and distilled water
was added for harvesting cells attached to the well
plate. Each sample (15 μl) was dispensed in a 96-
well plate, followed by the addition and incubation
of 60 μl of 1 mM para (p)-nitrophenyl-N-acetyl-
β-D-glucosaminide for 1 h. Carbonate buffer (150
μl, 0.05 M, pH 10) was added and the absorbance
at 405 nm was measured. All trials were conducted
in triplicate.

Histamine assay:

Cells were treated similar to when measuring
β-hexosaminidase. After incubation with the test
substance, the supernatant was collected and the cells
were collected and washed in Phosphate Buffered
Saline (PBS). Cells and supernatants were tested using
a histamine Enzyme-Linked Immunoassay (ELISA)
kit (Oxford Biomedical Research, Oxford, MI, USA)
following manufacturer’s guidelines. All trials were
conducted in triplicate.

Measurement of inflammatory mediators and
inflammatory cytokines:

RAW 264.7 cells were seeded at 4×10⁵ cells/well in a
6-well plate and cultured in a 37° CO₂ incubator for
24 h. Cells were incubated with Lipopolysaccharide
(LPS) (1 μl/ml) for 1 h and cultured for 24 h with the
test substance. Cell supernatants were collected and
Interleukin (IL)-1β, IL-6, Tumour Necrosis Factor alpha
(TNF-α) and Prostaglandin E2 (PGE2) were measured
by ELISA, following manufacturer’s guidelines (IL-1β,
IL-6, TNF-α ELISA kit; R&D Systems, Minneapolis,
MN, USA, PGE2 ELISA kit; Abcam, Cambridge, MA,
USA).

Animal experiment:

Animals: The animal study was approved by the
animal care and use committee at the Korea conformity
laboratories (IA16-00036). NC/NgaTndCrlj mice were
purchased from Orient Bio (Charles River Japan, 322,
Galmacho, Jungwon-gu, Sungnam, Korea). On the day
of receipt, a microbiological test report was received
and animal appearances were carefully examined.
Animals were maintained in a laboratory facility at
22°±3° and a relative humidity of 50 %±10 % under a
12:12 h light and dark cycle. Intensity of illumination
was LX 300. Noise level was 60 dB or less. During the
experimental period, the mice were fed a rodent diet
(Teklad Certified Irradiated Global 18 % protein rodent
diet, Harlan Co. Ltd., WI, USA) supplied by DooYeol
Biotech Co., Ltd. (107 Seongbo plaza, 91, Baumoe-ro,
Seocho-gu, Seoul, Korea). All animals were provided
with tap water purified by a reverse osmosis filtering
system ad libitum. All animals were acclimated for 6 d
after acquisition.

Induction of AD and grouping:

For the induction of AD, the hair on the back of the
animal was shaved using an electric razor and hair
removal cream on the 6^th d after arrival in the animal
facility. 2,4-Dinitrochlorobenzene (DNCB) (200 μl, 1
%) was applied to the skin for primary sensitization
and 2 d later, 2^nd induction was induced was induced
by the same method. 4 d after the second sensitization,
150 μl of 0.4 % DNCB was applied to the epidermal
skin to induce dermatitis (challenge stage) three times
a week for 6 w. Sensory evaluation was conducted
prior to initiation of administration and grouping was
performed by randomization based on the scores. Fifty
mice with AD were divided into 5 groups of 10 mice.
Test groups were administered with 800 (high-dose),
400 (mid-dose), 200 mg/kg body weight (low-dose)
concentrations and the effects were compared to those
in the AD control and positive groups. After induction
of AD, the test solution was administered by oral gavage
once a day, 7 d/w for 4 w, as shown in fig. 2.

Fig. 2: Schematic pictures of DNCB-applied NC/Nga mice of AD model and administration schedule of the test substance,

Clinical observation and weight measurement:

The general symptoms were observed immediately
after drug administration, once a day. Individual
records were maintained for each animal including
the mortality, type, date and grade of clinical signs.
Individual animal weight was recorded at acquisition,
grouping, before administration, necropsy once a week
during the study.

Sensory evaluations of skin lesions:

This evaluation method was used to compare scores by
the modified SCORing Atopic Dermatitis (SCORAD)
scoring system, which is a clinical visual evaluation
method generally used in AD. According to the index
of dermatitis scoring method, the degree of skin lesions
was divided into three categories scaling/dryness,
edema and excoriation/erosion. Scores were given
between 0 and 9, with 0 (no change), 1 (weak change),
2 (moderate change) and 3 (severe change) for each
item. Sensory evaluation was conducted on the 1^st d of
administration and once a week before necropsy.

Hematological test and serum IgE measurement:

On the day of autopsy, the animals were anesthetized
with isoflurane and blood samples were drawn from
the abdominal aorta and collected in Dipotassium
Ethylenediaminetetraacetic acid (EDTA-K2) tube
(Becton Dickinson, San Jose, CA, USA). The blood was
analysed using a haematology analyser (ADVIA 2120,
SIEMENS, Germany). White Blood Cell (WBC) count,
neutrophil, eosinophil, basophil, lymphocyte, monocyte
and large unstained cell counts, and the percentages of
neutrophils, eosinophils, basophils and lymphocytes
were analysed. The amount of IgE in blood serum
was measured using an IgE mouse ELISA kit (Abcam,
Cambridge, MA, USA. Lot No. GR3221697-1).

Histopathological examination:

Histopathological examination of skin tissue was
performed after autopsy. Hematoxylin and Eosin (HE) staining was performed to confirm the lesion in skin
tissue and mast cell and eosinophil infiltration was
confirmed by toluidine-blue and Congo red staining.
In HE staining, the lesion was classified as dermal
hyperplasia, dermal inflammation, epidermal erosion/
ulcer, hyperkeratosis and dermal fibrosis. The score was
given from 0 to 4 as follows 0 is normal, 1 is minimal,
2 is mild, 3 is moderate and 4 is severe. In toluidine
blue staining, the score was assigned, depending on
the distribution of mast cells as follows: 0: 0 to 10, 1:
10 to 30, 2: 30 to 70, 3: >70. In Congo red staining,
the score was given depending on the distribution of
eosinophils as follows: 0: 0, 1: <10, 2: 10-30, 3: 30-70,
4: >70. Mast cells and eosinophils were scored under
400× magnification.

Immunohistochemistry (IHC) of Protein Gene
Product (PGP) 9.5:

Sections (5 μm) were cut from all paraffin blocks
and mounted on glass slides. The sections were deparaffinized
by three immersions in xylene and then
hydrated by descending concentrations of ethanol (100
%, 90 % and 70 %) to PBS. After 1 h immersion in
hydrogen peroxide solution, the slides were then blocked
in goat serum and incubated overnight with mouse
PGP 9.5 antibody (Invitrogen, Waltham, MA, USA;
1:100). After washing, Dako Real Envision detection
system (K5007, Dako, Santa Clara, CA, USA) was
used to allow for quantitative detection neuroendocrine
marker PGP 9.5 respectively. The samples from each
section were evaluated at 40× magnification, using a
microscope (Olympus Provis AX-70; Olympus USA)
equipped with a camera (Olympus U-MAD 2, Olympus,
Japan). The images were captured using the program
MagnaFire 2.1B (Olympus).

Measurement of cytokine secretion in
splenocytes:

Spleens were aseptically harvested. Using the plunger
end of the syringe, mesh the spleen through the cell
strainer into a falcon tube. Strainer was rinsed several
times with serum free Roswell Park Memorial Institute
(RPMI) 1640 medium. Cells were centrifuged at 300 g
for 10 min at room temperature. The supernatant was
removed and cell pellet was suspended in Ammonium-
Chloride-Potassium (ACK) lysing buffer (A10492,
Invitrogen, Waltham, MA, USA) and taped for 15
min to remove the red blood cells. After washing
with serum free media, the cells were suspended in a
RPMI 1640 medium supplemented with penicillin 100 U/ml and streptomycin 50 μg/ml, 2-mercaptoethanol,
Non-Essential Amino Acid (NEAA) and FBS, 1 μg/
well anti-CD3 mAb (eBioscience, San Diego, CA,
USA) and 1 μg/well anti-CD28 mAb (ebioscience,
San Diego, CA, USA) were added and incubated for
48 h. After incubation, the cytokine released into the
culture medium was assayed using the Mouse IL-4
Quantikine (R&D systems, Minneapolis, MN, USA,
Lot No. P161144) and the mouse Interferon‐gamma
(IFN‐γ) Quantikine ELISA kits (R&D systems, Lot No.
P161140).

Statistical analysis:

All statistical analyses were performed using Statistical
Package for the Social Sciences (SPSS) 12.0 K program
(SPSS, Chicago, IL, USA). Numerical data was
calculated by mean±standard deviations. Differences
in means were considered statistically significant
when p<0.05. Statistical significance was represented
by percentage. The statistical differences between
vehicle and DNP control (in vitro)/vehicle and AD
control (in vivo) were examined using student’s t-test.
The statistical differences among test groups except
vehicle control were examined by standard one-way
Analysis of Variance (ANOVA). If the difference
was statistically significant, the data were analyzed by
parametric multiple comparison to evaluate the DNP
control. If the equal variance was admitted, Duncan’s
test was used and if the equal variance was not admitted,
Dunnett’s test was applied. In the hematological
analysis, one animal each from G2: AD control (0),
G3: AD+BSEWC (200), G5: AD+BSEWC (800) and
G6: AD+Ketotifen fumarate (1), was excluded from
the statistical analysis due to difficulty obtaining blood
sample. Seven individual samples were used for IgE
and cytokine analysis owing to lack of sample volume.

Results and Discussion

Copper is classified as a nutrient in functional health
foods, and is required for the transport and utilization
of iron and protection cells from harmful oxygen. Since
copper is an easy-to-analyse mineral, it was selected as
an indicator for quality control of the test solution. The
content of copper in egg white-chalcanthite, bamboo
salt and mixture of egg white-chalcanthite and bamboo
salt (1:20) was 19.48 %, 0 % and 0.98 %, respectively.

β-hexosaminidase secretion was measured as an
indicator for the inhibitory effect of degranulation,
which is an index of antigen-antibody reaction.
Comparative analysis of β-hexosaminidase released by cells showed that significantly elevated amounts
(p<0.01) in the DNP control compared to the negative
control. The amount of β-hexosaminidase released in
egg white-chalcanthite, bamboo salt, 1:15 and 1:20
mixture-treated groups was 42 %, 45 %, 46 % and 28 %,
respectively. This reduction was statistically significant
when compared to that of DNP control (p<0.01). The
positive control group showed a decrease of 29 % in
degranulation, which was similar to that of the 1:20
mixture treated group (p<0.01) (fig. 3A).

Fig. 3: Effect of bamboo salt, egg white-chalcanthite, bamboo salt with egg white-chalcanthite on IgE-induced degranulation in
RBL-2H3 cell line

The amount of histamine was measured as an indicator
of the inhibitory effect of degranulation which is an
index of the antigen-antibody reaction. The amount of
histamine within the cell as well as that secreted by the
cells was compared. DNP control showed 60 % histamine
release and negative control showed 34 % release from
the cells (p<0.01). The egg white-chalcanthite, bamboo
salt, 200 μg/ml 1:20 mixture treated groups showed 28
%, 37 % and 37 % histamine release, respectively. It
was a statistically significant decrease when compared
to that of DNP control (p<0.01) (fig. 3B).

Significantly elevated levels of IL-1β were observed in the LPS control group compared to the vehicle
control group. However, incubation with 10 μg/ml
egg white-chalcanthite; 200 μg/ml bamboo salt; 1:10,
1:15 and 1:20 egg white-chalcanthite and bamboo salt
mixture-treated groups showed reduced levels of IL-1β,
suggesting a reduction in inflammation (p<0.01) (fig.
4A). Similarly, elevated expression of IL-6 was in the
LPS control group was significantly attenuated in 200
μg/ml 1:10, 1:15 and 1:20 egg white-chalcanthite and
bamboo salt mixture-treated groups compared to the
LPS control group (p<0.01) (fig. 4B). The expression
of TNF-α, although increased in the LPS control group
compared to the vehicle control group, was significantly
decreased in 200 μg/ml bamboo salt, 1:10, 1:20 egg
white-chalcanthite and bamboo salt mixture-treated
groups compared to the LPS control group (p<0.01)
(fig. 4C). The expression of PGE2 was increased in
the LPS control group compared to the vehicle control
group, but was reduced in all test substance-treated
groups compared to LPS control group (p<0.01) (fig.
4D). In particular, 1:20 egg white-chalcanthite and
bamboo salt mixture effectively reduced the release of
inflammatory cytokines and mediators in all test items.

Fig. 4: Effect of bamboo salt, egg white-chalcanthite, bamboo salt with egg white-chalcanthite on the expression of inflammatory
mediator and cytokines

No death or other abnormalities were observed except
for AD in the AD-induced animal group during the
experimental period. Weight-measurement results
indicated a reduction in 1 w body weight of ADinduced
group compared to the normal control group
(p<0.05). However, this change was temporary and a normal weight gain was observed thereafter (fig. 5A).
Skin sensory evaluation results indicated a significant
reduction in the AD-induced group compared to the ADinduced
control group at 3 and 4 w after administration
of 800 mg/kg/d (p<0.01 or p<0.05) (fig. 5B).

Fig. 5: Clinical skin features of DNCB-applied NC/Nga mice after repeated 4 w oral administration of the test substances

Hematological tests showed that WBC in 800 mg/kg/d
treated group and monocyte levels in 800 mg/kg/d and
ketotifen fumarate-treated groups were statistically
higher than AD induced excipient control group
(p<0.05 or p<0.01). No statistically significant changes
were observed in other test items compared to AD
control group (fig. 6A). The blood IgE concentration
did not show statistically significant changes in the
test substance administration group, but the blood IgE
concentration in the test substance-treated group and the positive control substance were decreased compared to
that in the AD-induced control group (fig. 7A). IL-4
level was significantly decreased in splenocytes from
the group treated with 400 mg/kg bamboo salt with egg
white-chalcanthite (p<0.01) (fig. 7B). In the 800 mg/
kg and ketotifen fumarate-treated groups, IFN-γ level
was reduced (fig. 7C) and the IL-4/IFN-γ ratio was
significantly increased compared to in the AD control
group (fig. 7D).

Fig. 6: Clinical observation of DNCB-applied NC/Nga mice after repeated 4 w oral administration of the test substances,

Fig. 7: Hematological analysis on solenocytes of DNCB-applied NC/Nga mice after repeated 4 w oral administration of the test
substances

The vehicle control did not show any abnormality in
terms of lesions observed in the skin tissue and all
items were scored as 0. AD was observed in AD control
mice with the highest scores in dermal hyperplasia and
inflammation confirming robust induction of AD. In the
200, 400 and 800 mg/kg 1:20 mixture of egg whitechalcanthite
and bamboo salt-treated group, the score
of dermal hyperplasia and inflammation decreased in a
dose-dependent manner. Particularly in the 800 mg/kg
treated group, the scores of hyperkeratosis and dermal
fibrosis decreased simultaneously. The AD control
group got the highest score in the sum of each parameter
and the score was reduced in the test substance-treated
group in a dose-dependent manner. However, there was
no significant difference in epidermal erosion/ulcer
score among the groups. The positive control group
showed a similar score to the 800 mg/kg treated-group.

The AD control group exhibited the highest mast cell
counts while the vehicle group had a score of 0. In the
200, 400 and 800 mg/kg 1:20 mixture of egg whitechalcanthite and bamboo salt-treated groups, the score
decreased in a dose-dependent manner. The positive
control group got the lowest score. The vehicle control
group was awarded a score of 0 in terms of eosinophil
counts. The scores of 200, 400, and 800 mg/kg 1:20
mixture of egg white-chalcanthite and bamboo salttreated
groups were reduced in a dose-dependent
manner because the AD control group did not show the
highest score. The positive control group received the
same score as the 800 mg/kg-treated group (fig. 8).

Fig. 8: Histopathological findings of dorsal back skin of DNCB-applied NC/Nga mice after repeated 4 w oral administration of the
test substances

PGP 9.5 was immunohistochemically stained to confirm
the degree of nerve fiber infiltration. IHC results
showed high nerve fiber infiltration in AD control group
skin tissue whereas infiltration was barely observed in
the vehicle control group. The degree of nerve fiber
infiltration in bamboo salt with egg white-chalcanthitetreated
group was lowered in a dose-dependent manner.
The positive control group showed similar degree of
invasion as the 800 mg/kg bamboo salt with egg whitechalcanthite-
treated group (Fig. 9).

Fig. 9: PGP 9.5 IHC of dorsal back skin of DNCB-applied NC/Nga mice after repeated 4 w oral administration of the test substances

Based on the above results, it was found that when the
test substance, bamboo salt with egg white-chalcanthite,
was orally administered to the AD model for 4 w
repeatedly, the dermatitis decreased visually, dermal
hyperplasia/inflammation, mast cell/eosinophils counts
and degree of nerve fiber infiltration were decreased
according to histopathological examination. In
addition, the secretion rate of IFN-γ in high-dose treated
group was lower than that in AD control group. And,
bamboo salt and egg white-chalcanthite help to relieve
symptoms of AD in vitro as well. We believe that the
results of this study will encourage further research on
the anti-inflammatory effect of the combination of egg
white-chalcanthite and bamboo salt to develop novel
therapeutic agents to treat widespread dermatological
conditions such as AD.

Egg white-chalcanthite and bamboo salt mixture is a
medicinal product that has been traditionally used to
treat AD in Hamyang, Korea. One of the ingredients,
chalcanthite is a mineral medicinal herb that contains
naturally occurring copper vitriol, and is described as
“stone gall” in Shennong’s Materia Medica, Shen nong ben cao jing. The major components of the chalcanthite
are Cupric oxide, carbonate and water whereas zinc,
gallium, titanium, sodium, calcium, iron, aluminum,
magnesium, silicon, chromium and nickel are minor
constituents[11,12]. The efficacy of chalcanthite as a single
medicinal product is presumed to be lowered in toxicity
and higher in absorption effect when administered after
processing with egg white. Combination with egg
white can prevent liver dysfunction as well as disorders
of the basal ganglia of brain and kidneys, when copper
is consumed for prolonged periods of time[13]. Despite
its diverse medicinal applications, research studies on
chalcanthite have not been conducted while studies
on mineral medicine have been quite limited. This is
due to a general lack of concern about the toxicity of
mineral medicines and an overall lack of systematic
pharmacological analysis. Mineral medicines have
been passed down through thousands of years of history
alongside botanical and animal derived medicines[14,15].
If the active component and efficacy of medicinal
minerals are identified in a scientific way, this may
contribute to developing more effective novel drug medicines.

The other ingredient, bamboo salt, is a traditionally
used medicinal ingredient, described as salt in various
pamphlets such as in the traditional book, Donguibogam.
However, it is not specifically referred to as bamboo
salt. It is believed that though historically it has existed
in multiple forms, the name bamboo salt itself was used
since 1981[16]. Currently, people believe that one of the
causes of hypertension is excessive sodium intake and as
a result, salt consumption as a medicine may be viewed
with some concern. Minerals are essential for human
activity and are often obtained in adequate amounts
through salt consumption[17]. The most commercially
produced salt is NaCl. However, it is known that CaCl₂,
magnesium, potassium, selenium and germanium
are relatively high in natural sun dried-salt, salt from
vegetable (Salicornia europaea L.) or bamboo salt[18].
In particular, manufacturers frequently claim bamboo
salt to be devoid of all the bad ingredients of refined
salt that contains a significant amount of minerals and
trace elements such as magnesium and calcium, which
are beneficial to the human body[19]. Devoid of toxicity
and with increased concentration of minerals, bamboo
salt may be of significant therapeutic value. There are
scientific explanations for the relief from inflammation
in mineral AD as follows, Yoou et al.[20] reported that
skin inflammation releaved by oral administration of
bamboo salt in atopic dermititis mouse model. Kim et
al.[21] reported that magnesium-rich marine minerals
could be used as an adjunctive therapy, and Yoon et al.[22] reported that selenium-rich hot spring water has
an anti-inflammatory and antimicrobial effect in chronic
bacterial prostatitis rat. Further studies are required to
elucidate the relationship between the mineral content
in bamboo salt and dermatitis.

Recent research has shown that bamboo salt with
egg white-chalcanthite has tremendous potential as a
therapeutic agent in multiple diseases. It is reported
that bamboo salt with egg white-chalcanthite can be
developed and used as an effective therapeutic agent
having anticancer effect in lung cancer[23], therapeutic
effect in arthritis[23-25], and induction of apoptotic cell
death in vitro[26]. Since egg white-chalcanthite and
bamboo salt mixture exhibits various efficacy, toxicity
test results using various routes of administration are
essential for widespread use in clinical practice. Singledose,
repeated-dose, sensitization, skin irritation, and
genotoxicity tests are necessary and the side effects of
administration should be closely examined.

Acknowledgements:

This work was supported by the technology
development program (No. S2424212) funded by the
Ministry of SMEs and Startups (MSS, Korea). Somin
Lee, Hyeon Yeol Ryu and Eun A Choi conceived the
idea and designed the experiments. Somin Lee, Hyeon
Yeol Ryu, Su Jin Kang and Hye Jin Kim performed the
experiments. Eun A Choi, Hong Geun Kim, Hyeong Ho
Seo, Ji Young Choi contributed materials and reagents.
Somin Lee analysed the data and wrote the manuscript.
Kyung Seuk Song and Chanhee Chea reviewed the
manuscript.

Conflict of interest:

The authors have no conflicts of interest to declare.

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